Monoclonal antibodies reactive with defined regions of the T cell antigen receptor

ABSTRACT

The present invention relates to monoclonal antibodies which recognize defined regions of the T-cell receptor (TCR). In a specific embodiment, the invention provides monoclonal antibodies which are reactive with a constant region of the alpha chain of the TCR. In particular embodiments, the invention relates to two monoclonal antibodies, termed αF1 and αF2, which react with two different epitopes on the framework region of the α monomer of the TCR molecule. In another specific embodiment, the invention is directed to monoclonal antibodies reactive with a variable region of the beta chain of the TCR. In particular, the invention provides two monoclonal antibodies, termed W112 and 2D1, which react with β chain variable regions Vβ5.3 and Vβ8.1, respectively. In another specific embodiment, the invention is directed to monoclonal antibodies reactive with a variable region of the delta chain of the TCR. In particular, the invention provides monoclonal antibody δTCS1, isotype IgG2a. The monoclonal antibodies of the invention have value in diagnosis and therapy and are useful tools for study of the immune system.

This is a division of U.S. application Ser. No. 08/083,408, filed Jun.25, 1993, which is a division of U.S. application Ser. No. 07/449,692,filed Dec. 11, 1989 and now U.S. Pat. No. 5,223,426, which is a CIP ofU.S. application Ser. No. 07/343,189, filed Apr. 25, 1989 and nowabandoned, which is a CIP of U.S. application Ser. No. 07/284,511, filedDec. 15, 1988 and now abandoned.

1. INTRODUCTION

The present invention is directed to monoclonal antibodies, whichrecognize defined regions of the T cell antigen receptor. The monoclonalantibodies of the invention have value in diagnosis and therapy and areuseful tools for study of the immune system.

2. BACKGROUND OF THE INVENTION

2.1. THE T CELL ANTIGEN RECEPTOR

T lymphocytes interact with antigens through the T cell antigen receptor(TCR) complex. The TCR is a clone-specific heterodimer on T cells, whichrecognizes its target antigen in association with a majorhistocompatiblity antigen. The TCR has been shown to be noncovalentlyassociated with the CD3 complex. TCR is highly polymorphic between Tcells of different specificities. Approximately 90 percent of peripheralblood T cells express a TCR consisting of an α polypeptide and a βpolypeptide. A small percentage of T cells have been shown to express aTCR consisting of a γ polypeptide and a δ polypeptide. (Regarding TCRmolecules, see Davis and Bjorkman, 1988, Nature 334:395-402; Marrack andKappler, 1986, Sci. Amer. 254: 36; Meuer et al., 1984, Ann. Rev.Immunol. 2:23-50; Brenner et al., 1986, Nature 322:145-159; Krangel etal., 1987, Science 237:1051-1055; Hata et al., 1987, Science238:678-682; Hochstenbach et al., 1988, J. Exp. Med. 168:761-776). Thechains of the T cell antigen receptor of a T cell clone are eachcomposed of a unique combination of domains designated variable (V),[diversity (D),] joining (J), and constant (C) (Siu et al., 1984, Cell37:393; Yanagi et al., 1985, Proc. Natl. Acad. Sci. USA 82:3430).Hypervariable regions have been identified (Patten et al., 1984, Nature312:40; Becker et al., 1985, Nature 317:430). In each T cell clone, thecombination of V, D and J domains of both the alpha and the beta chainsor of both the delta and gamma chains participates in antigenrecognition in a manner which is uniquely characteristic of that T cellclone and defines a unique binding site, also known as the idiotype ofthe T cell clone. In contrast, the C domain does not participate inantigen binding.

2.2. T CELL ANTIGEN RECEPTOR GENES

TCR genes, like immunoglobulin genes, consist of regions which rearrangeduring T cell ontogeny (Chien et al., 1984, Nature 312:31-35; Hedrick etal., 1984, Nature 308:149-153; Yanagi et al., 1984, Nature 308:145-149).In genomic DNA, each TCR gene has V, J, and C regions; TCR β and δpolypeptides also have D regions. The V (variable), D (diversity), J(junctional) and C (constant) regions are separated from one another byspacer regions in the DNA. There are usually many variable regionsegments and somewhat fewer diversity, junctional, and constant regionsegments. As a lymphocyte matures, these various segments are splicedtogether to create a continuous gene sequence consisting of one V, (D),J, and C region. TCR diversity, and thereby T cell specificity, isderived from several sources (Barth et al., 1985, Nature 316:517-523;Fink et al., 1986, Nature 321:219-225): a multiplicity of germline genesegments (Chien et al., 1984, Nature 309:322-326; Malissen et al., 1984,Cell 37:1101-1110; Gascoigne et al., 1984, Nature 310:387-391; Kavaleret al., 1984, Nature 310: 421-423; Siu et al., 1984, Nature 311:344-349;Patten et al., 1984, Nature 312:40-46), combinatorial diversity throughthe assembly of different V, D, J, and C segments (Siu et al., 1984,Cell 37:393-401; Goverman et al., 1985, Cell 40:859-867), and junctionalflexibility, N-region diversity and the use of either multiple D regionsor any of the three translational reading frames for Dβ segments. TCRdiversity does not appear to arise from the somatic hypermutationmechanism observed for immunoglobulins (Barth et al., supra). As aresult of these mechanisms, TCRs are generated which differ in theiramino-terminal, or N-terminal, domains (called variable, or V regions,constructed from combinations of V, D, and J gene segments) but are thesame elsewhere, including their carboxy-terminal, or C-terminal domains(called constant regions). Accordingly, an almost limitless repertoireof TCR is established.

The Vβ gene of the TCR appears to resemble most closely theimmunoglobulin V gene in that it has three gene segments, Vβ, Dβ, andJβ, which rearrange to form a contiguous Vβ gene (Siu et al., 1984, Cell37:393-401). The β locus has been well characterized in mice, where itspans 700-800 kilobases of DNA and is comprised of two nearly identicalC regions tandemly arranged with one D element and a cluster of 5-6 Jelements 5' to each (Kronenberg et al., 1986, Ann. Rev. Immunol.3:537-560). Approximately twenty to thirty Vβ regions are locatedupstream (5') to the D, J, and C elements (Behlke et al., 1985, Science229:566-570) although Vβ genes may also be located 3' to the murine Cβgenes (Malissen et al., 1986, Nature 319:28). Study of the structure anddiversity of the human TCR β-chain variable region genes has led to thegrouping of genes into distinct Vβ subfamilies (Tillinghast et al.,1986, Science 233:879-883; Concannon et al., 1986, Proc. Natl. Acad.Sci. USA 83:6598-6602; Borst et al., 1987, J. Immunol. 139:1952-1959).

The γTCR gene was identified, first in mice (Saito et al., 1984, Nature309:757-762; Kranz et al., 1985, Nature 313:762-755; Hayday et al.,1985, Cell 40:259-269) and then in humans (Lefranc et al., 1985, Nature316:464-466; Murre et al., 1985, Nature 316:549-552). The human γTCRlocus appears to consist of between five and ten variable, five joining,and two constant region genes (Dialynas et al., 1986, Proc. Natl. Acad.Sci. U.S.A. 83:2619).

The TCR α and δ locus are next to one another on human chromosome 14.Many TCR δ coding segments are located entirely within the α gene locus(Satyanarayana et al., 1988, Proc. Natl. Acad. Sci. USA 85:8166-8170Chien et al., 1987, Nature 330:722-727; Elliot et al., 1988, Nature331:627-631). It is estimated that there are a minimum of 45-50 Vαregions (Becker et al., Nature 317:430-434) whereas there are onlyapproximately 10 Vδ regions (Chien et al., 1987, supra). In peripheralblood, two predominant Vδ genes appear to be expressed, namely, Vδ1 andVδ2, identifiable by monoclonal antibodies, δTCS1 and BB3, respectively.Nucleic acid sequences of TCR α genes have been reported (Sim et al.,1984, Nature 312:771-775; Yanagi et al., 1985, Proc. Natl. Acad. Sci.USA 82:3430-3434; Berkout et al., 1988, Nucl. Acids Res. 16:5208).

2.3. ANTIBODIES TO THE T CELL ANTIGEN RECEPTOR

Clonotypic antibodies react only with a particular clone of T cells.Acuto et al. produced clonotypic monoclonal antibodies against a humanthymocyte cell line, and thereby identified the TCR in relativelyundifferentiated T3⁺ cells (1983, Cell 34:717-726). Meuer et al. showedthat anti-TCR clonotypic monoclonal antibodies coupled to sepharosebeads could induce production of interleukin-2 (1984, Proc. Natl. Acad.Sci. 81:1509-1513). Anti-TCR clonotypic antibody directed toward the CT8cell line could only block cytotoxic effector cell function of that Tcell line (Meuer et al., 1984, Ann. Rev. Immunol. 2:23-50). Antibodieswhich recognize TCR from many T cell lines recognize shared epitopes, orframework regions, of TCR peptides. Brenner et al. found that differentcloned T cell lines shared antigenic determinants, none of whichappeared to be accessible at the cell surface (1984, J. Exp. Med.160:541-551). β-Framework-1 (βF1) monoclonal antibody reacts with a"hidden determinant" on the surface of viable T cells, and recognizesthe TCR β polypeptide in Western blots (Brenner et al., 1987, J.Immunol. 138:1502-1509). Another framework antibody, WT31, originallythought to be a framework reagent is useful in cell binding, but isinefficient in immunoprecipitation studies (Spits et al., 1985, J.Immunol. 135:1922-1928). WT31 now appears to recognize a CD3determinant.

2.4. RHEUMATOID ARTHRITIS

Rheumatoid arthritis (RA), a chronic, recurrent, inflammatory diseaseprimarily involving joints, affects 1-3% of North Americans, with afemale to male ratio of 3:1. Severe RA patients tend to exhibitextra-articular manifestations including vasculitis, muscle atrophy,subcutaneous nodules, lymphadenopathy, splenomegaly and leukopenia.Spontaneous remission may occur; other patients have brief episodes ofacute arthritis with longer periods of low-grade activity; still othersprogress to severe deformity of joints. In some patients with rheumatoidarthritis, particularly those with long-standing disease, aconstellation of symptoms called "Felty's syndrome" develops, in whichthe typical arthropathy is accompanied by splenomegaly and neutropenia.It is estimated that about 15% of RA patients (severe RA and Felty'ssyndrome) become completely incapacitated ("Primer on the RheumaticDiseases, 8th edition, 1983, Rodman, G. P. & Schumacher, H. R., Eds.,Zvaifler, N. J., Assoc. Ed., Arthritis Foundations, Atlanta, Ga.).

The antigenic stimulus initiating the immune response and consequentinflammation is unknown. Certain HLA types (DR4, Dw4, Dw14 and DR1) havean increased prevalence of RA, perhaps leading to a geneticsusceptibility to an unidentified factor which initiates the diseaseprocess. The association with DR4 is highest for Felty's Disease andsevere RA (Westedt, M. L., et al., Annals of Rheumatic Diseases, 1986,45, 534-538). Relationships between Epstein Barr virus and RA have beensuggested. Synovial lymphocytes produce IgG that is recognized asforeign and stimulates a local immune response with production ofanti-IgG-antibodies (rheumatoid factors). Immune complexes are formed byactivation of the complement system which results in inflammationincluding activation of lysozyme and other enzymes. Helper T cellinfiltration of the synovium and liberation of lymphokines such as IL6lead to further accumulation of macrophages and slowly progressing jointdestruction (erosions).

The approach to drug treatment in rheumatoid arthritis has beendescribed as a pyramid ("Primer on the Rheumatic Diseases", supra).First line agents include aspirin and NSAIDS (non-steroidalanti-inflammatory drugs). When these agents fail, gold salts,penicillamine, methotrexate, or antimalarials, known as conventionalsecond line drugs, are considered. Finally, steroids or cytotoxics aretried in patients with serious active disease that is refractory tofirst and second line treatment. Cyclosporine is now suggested to have arole in the treatment of patients whose disease is unresponsive toaspirin, NSAIDS, gold or penicillamine. However, the currentexperimental drugs to treat severe RA patients may prove too toxic evenif they are effective.

Numerous efforts have been directed to developing safer and moreefficacious immunotherapy to replace these toxic drugs. Severe RApatients who were treated with total lymphoid irradiation or thoraicduct drainage experienced significant improvement of disease symptoms.These procedures are not suitable for routine application. Due to theseencouraging findings, however, and to the demonstration of the presenceof T cells in the synovial infiltrate, it is possible to design newimmunotherapies to specifically eliminate T cells. Most of these newexperimental immunotherapies are targeted toward all or the bulk of Tcells, and thus may produce significant side effects. A better approachfor selective immunotherapy may be to eliminate only the smallproportion of T cells that are involved in RA.

2.5. ROLE OF T CELLS IN RHEUMATOID ARTHRITIS

Evidence has accumulated supporting a role for T-cells in thepathogenesis of rheumatoid arthritis (RA). The synovial tissue andsurrounding synovial fluid of patients with rheumatoid arthritis (RA)are infiltrated with large numbers of cells. Activated and resting Tcells can mediate tissue damage by a variety of mechanisms including thedirect cytotoxicity of target cells expressing specific antigen incombination with the appropriate HLA restricting elements. The strongassociation of certain HLA products with RA has led researchers toimplicate T cells in the autoimmune destruction of RA patient joints. Infact, HLA DR4, Dw4 and Dw14 gene products are among the major class IImolecules that contribute significantly to disease susceptibility in RApatients (Nepom, B., et al., 1987, Abstracts of Amer. Rheumatism Assoc.,p. S25; Todd, J. A., et al., 1988, Science 240:1003-1009), and they arecapable of restricting antigen recognition of CD4+ T cells, primarily.Other autoimmune diseases also show a high correlation between diseasesusceptibility and HLA expression (Table 1).

This genetic basis of disease risk has resulted in phenotypic analysisof the T cells found within diseased joints. Previously, comparisons ofT cells from RA joints and RA peripheral blood (PB) demonstratedsignificant differences in CD4 or CD8 phenotype, therefore implying aselection of T cells involved in disease activity. Most studies agreethat synovial tissue-infiltrated T cells were mostly CD4+ helper-inducer(4B4+) cells (Duke, O., et al., 1987, Arth. Rheum., 30, 849) while thePB usually contained a mixture of CD4+ and CD8+ cells including bothhelper-inducer cells and suppressor-inducer cells (2H4+) (Emery, P., etal., 1987, Arth. Rheum., 30, 849). In contrast, there is additionalevidence that the CD4+ infiltrate may be predominantlysuppressor-inducer cells (2H4+) (Mikasaka, N., et al., 1987, Amer.Rheum. Abstracts, p. S39).

2.6. γδ POSITIVE T CELLS

γδ TCR may be the principal TCR in selected sites such as the skin orother organs. Although the function of the γδ positive T cells islargely unknown, they appear to be involved in non-MHC-restrictedcytotoxicity and IFN-γ production. γδ⁺ T cells are known to secrete avariety of lymphokines, such as TNF alpha, IL2 and IL4 (Bluestone, J. A.and Matis, L. A., 1989, J. Immunol. 142, 1785-1788). The totalpopulation of δ+ T cells can be identified by the monoclonal antibody,TCRδ1, which recognizes a major framework determinant on the δ TCR(Band, H., et al., 1987, Science, 238, 682). A subset of γδ positiveT-lymphocytes can be identified by the monoclonal antibodies, δTCS1(anti-V.sub.δ 1; Wu, Y-J., et al., 1988, J. Immunol. 141, 1476-1479) andBB3 (anti-V.sub.δ TCR, Bottino, C., et al., 1988, J. Exp. Med., ˜˜,491-505). A study by Grossi, C. E., et al. (Proc. Natl. Acad. Sci.,1989) indicated that δTCS1⁺ T cells exhibit motile cell morphology andmigrate in tissue culture. δTCS1⁺ T cells were also shown to be potentkiller T cells (Rivas, A., et al., 1989, J. Immunol., 142, 1840-1846).

3. SUMMARY OF THE INVENTION

The present invention is directed to monoclonal antibodies whichrecognize defined regions of the T cell antigen receptor (TCR). Theantibodies of the invention bind to epitopes of the variable, diversity,joining, and/or constant regions of the alpha, beta, gamma, or deltachains of the T cell antigen receptor.

In a specific embodiment, the invention provides monoclonal antibodieswhich are reactive with a constant region of the alpha chain of the TCR.In particular embodiments, the invention relates to the two monoclonalantibodies, termed αF1 and αF2, which react with two different epitopeson the framework, or constant, region of the α monomer of the TCRmolecule. In various embodiments of the invention, αF1 or αF2, or both,or fragments or derivatives thereof, can be used to bind to the a TCRconstant region amino acid sequences, either as part of an intact TCRcomplex or α peptide, or a fragment thereof.

In another specific embodiment, the invention is directed to monoclonalantibodies reactive with a variable region of the beta chain of the TCR.In a preferred embodiment of the invention, the monoclonal antibodiesreact with a "minor framework" region of the TCR beta chain, and therebyrecognize a subpopulation of T cells. In particular, the inventionprovides two monoclonal antibodies, termed W112 and 2D1, which reactwith β-chain variable regions Vβ5.3 and Vβ8.1, respectively, and therebyrecognize between 0.3 to 5% and 0.5 to 13% of peripheral bloodlymphocytes, respectively. In various embodiments of the invention, W112or 2D1, or fragments or derivatives thereof, can be used to bind withβTCR variable region amino acid sequences, either as part of an intactTCR or peptide, or T cell-surface molecule, or a fragment thereof.

In another specific embodiment, the invention is directed to monoclonalantibodies reactive with a variable region of the delta chain of theTCR. In a preferred embodiment of the invention, the monoclonalantibodies react with the Vδ1 region of the TCR delta chain, and therebyrecognize a subpopulation of T cells.

In a further specific embodiment, the invention is directed to aparticular monoclonal antibody, δTCS1, which is of the IgG2a isotype.

The monoclonal antibodies of the invention have value in the diagnosisand therapy of conditions and diseases affecting the immune system.

In particular embodiments of the invention, rheumatoid arthritis orFelty's syndrome may be diagnosed by detecting increased percentages oftotal T cells which express certain delta or beta chain T cell receptorvariable region genes in a patient sample. In specific embodiments ofthe invention, rheumatoid arthritis may be diagnosed by detectingincreased percentages of total T cells which express Vδ1, Vβ3, Vβ9, orVβ10 T cell receptor variable regions in a patient sample. In apreferred embodiment of the invention, rheumatoid arthritis may bediagnosed by detecting increased percentages of total T cells which areδTCS1 positive in a patient sample.

In further particular embodiments of the invention, rheumatoid arthritismay be treated by administering a therapeutically effective amount of amonoclonal antibody, or fragment or derivative thereof, which recognizesan epitope of the variable region of the beta chain or the delta chainof a T cell antigen receptor. According to specific embodiments,monoclonal antibodies which recognize epitopes of Vδ1, Vβ3, Vβ9, or Vβ10variable regions of the T cell antigen receptor may be used to treatrheumatoid arthritis.

The invention also provides for therapeutic compositions comprising themonoclonal antibodies of the invention.

3.1. ABBREVIATIONS AND DEFINITIONS

As used herein, the following terms will have the meanings indicated:

C=constant

D=diversity

ELISA=enzyme linked immunosorbent assay

J=joining

mAb=monoclonal antibody

PBL=peripheral blood lymphocytes

PMA=phorbol 12-myristate 13-acetate

SDS-PAGE=sodium dodecylsulfate polyacrylamide gel electrophoresis

TCR=T cell antigen receptor

V=variable

anti-clonotypic antibody=an antibody that reacts solely with the T cellclone against which it was raised. Also referred to as an anti-idiotypicantibody.

anti-minor framework antibody=an antibody that reacts with a minorframework determinant present on a subset of T cells. Anti-minorframework antibodies recognize small percentages of PBLs, i.e., lessthan 20% in a normal subject. Anti-minor framework antibodies can beused to define closely related TCRs or TCR families.

anti-major framework antibody=an antibody that reacts with a majorframework determinant present on a large population of T cells.Anti-major framework antibodies will recognize at least 20% of PBLs in anormal subject.

RES=reticuloendothelial system.

RA=Rheumatoid Arthritis

PB=peripheral blood

ST-line=RA synovial tissue-derived T cells

PB-T=peripheral blood-derived T cells

FS=Felty's Syndrome

SSA=Seronegative Spondyloarthropathies

EBV=Epstein-Barr virus

PBS=phosphate buffered saline

NK=natural killer

NST-line=non-RA synovial tissue-derived T cells

HLA=human leukocyte antigen

4. DESCRIPTION OF THE FIGURES

FIG. 1. Immunoprecipitation of ¹²⁵ I labeled HPB-cell lysates andMolt-13 cell lysates. HPB- and Molt-13 cells were surface labeled with¹²⁵ I and lysed in 1% NP-40. Cell lysates were incubated with mAb andthe precipitated immune complex was applied to 10% SDS-PAGE.Electrophoresis was run under non-reducing (A) and reducing conditions(B). The precipitated TCR proteins were detected by autoradiography.

FIG. 2. TCR immunoprecipitation from Jurkat cells and PBL with αF1 andαF2. ¹²⁵ I labeled Jurkat cells and PBL were lysed in 1% NP-40 andincubated with αF1 and αF2 and control mAb. The immune complexprecipitated was revealed by 10% SDS-PAGE under non-reducing conditions.

βF1 antibody was a positive control for immunoprecipitating the αβTCR.δTCS1 antibody reacts with δγ⁺ cells, not ab⁺ cells.

FIG. 3. Immunoperoxidase staining of human thymus and tonsilar tissue. Aand B are thymus cortex; C and D are thymus medulla; E and F are humantonsil. βF1 stains about 70% of cells in cortex (A), over 90% in medulla(C) and all T cells in the interfollicular region of tonsil (E). Asimilar staining pattern is observed when αF1 is used (B, D, and F).Note that αF1 stains less thymus cortex cells (B).

FIG. 4. Competition assay of TCRα peptides with HPB-TCR. Two-folddilutions of free α141-159 (amino acids 141-159 of TCRα)(open circle) orα212-231 (amino acids 212-231 of TCRα)(solid circle) were mixed with 10%HPB-lysates and incubated with αF1 (a) or αF2 (b) in a TCR-ELISA assay.Peptide concentration is shown on the X-axis in logarithmic scale. αF1and αF2 were used at 4 μg/ml.

FIG. 5. Immunoprecipitation of in vitro translated α chain T cellantigen receptor protein with mAb αF1 and αF2. Aliquots of proteinmixture containing both α chain and rabbit globin (lane 6) wereimmunoprecipitated separately with anti-Cα antibodies, αF1 (lane 1) andαF2 (lane 2), and isotype matched, irrelevant antibodies in lane 3 andlane 4. Lane 5, in vitro translation in the absence of any exogenousRNA. Samples were analyzed on a 12.5% SDS-PAGE which was dried andsubsequently autoradiographed.

FIG. 6. A. A schematic of the hybridoma screening ELISA. Microtiterplate wells were coated with goat anti-mouse Ig Fc and washed to removeunbound reagent. Non-specific sites were blocked with bovine serumalbumin. Aliquots of hybridoma supernatants were added to the wells withblocking buffer, incubated, and followed by washing to remove unboundproteins. NP40 generated cell lysates from αβ TCR cell lines were added,wells were washed, and biotin-conjugated βF1 F(ab)₂ was added. Afterwashing to remove unbound reagent, HRP conjugated streptavidin wasadded, wells were washed, and color was developed using an HRPsubstrate, o-phenylenediamine.

B. Comodulation assay: Jurkat cells were incubated with antibodiesovernight, washed, incubated with FITC-labeled OKT3 and analyzed by flowcytometry. Antibody 2D1 caused a dramatic reduction (over 90%) of OKT3surface staining. C305, an anti-Jurkat IgM isotype antibody and OKT3also modulated the CD3 expression, while isotype-matched controlantibody W4 did not. This data indicated that the epitope recognized by2D1 is on the CD3-TCR complex.

FIG. 7. A. Immunoprecipitation of HPB with W112 and control antibodiesunder non-reducing conditions followed by SDS-PAGE under reducingconditions. W112 and βF1 precipitated the αβ TCR heterodimer of 48 kD(α) and 40 kD (β). Normal mouse serum did not precipitate the αβ TCRheterodimer.

B. Immunoprecipitation of Jurkat cell lysates with 2D1 and controlantibodies under non-reducing conditions followed by SDS-PAGE underreducing conditions. 2D1 precipitated the α,β TCR heterodimer of 48 kD(α) and 40 kD (β). βF1 and αF1 precipitated the same 48 kD and 40 kDbands, while negative control antibody δTCS1 did not.

FIG. 8. Western blot analysis using HPB cell membranes. W112 recognizedthe 40 kD TCR β chain protein as indicated by the arrow. βF1 alsodetected the 40 kD band. Normal mouse serum did not react with thisband. αF1 detected a different band of a higher molecular weight.

FIG. 9. Immunoprecipitation of metabolically labelled Jurkat celllysates. 2D1 precipitated the 40 kD β chain TCR, as did βF1.Isotype-matched negative control antibody LL-112 was unreactive.

FIG. 10. Flow cytometric analysis of peripheral blood lymphocytes (PBLs)from one normal donor using W112 and 2D1 antibodies. The positivecontrol pan-T monoclonal antibody OKT3 stained 70% of the PBLs. W112stained a small subpopulation consisting of 3% of the PBLs and 2D1stained a small subpopulation of 5.5% PBLs. Normal mouse serum did notreact with any of the PBLs.

FIG. 11. Levels of TCRδ1 bearing T cells (left panel) and δTCS1 bearingT cell (right panel) in the PB of patients with RA, FS and NML.Arithmetic mean values are represented by the `X` and the error barsrepresent ±1 standard error (S.E.).

FIG. 12. Ratio of δTCS1/TCRδ1 bearing T cells in patients with RA, FSand NML. Notations as per FIG. 11.

FIG. 13. Relationship between the percent of TCRδ1 T cells and thepercent of δTCS1 T cells in patients with FS.

FIG. 14. Levels of TCRδ1 bearing T cells (upper panel) and δTCS1 bearingT cells (lower panel) in the PB and SF of RA and SSA patients. Notationsas per FIG. 11.

FIG. 15. Ratio of δTCS1/TCRδ1 bearing T cells in the PB and SF of RApatients and SF of patients with SSA. Notations as per FIG. 11.

FIG. 16. Synovial derived T cell (ST-13) cytotoxicity of type A synovialtarget cells and the cold target inhibition by K562. Effector to ⁵¹ Crlabelled target cell ratio was 1:1 while increasing concentrations ofunlabelled target cells were added. The standard deviations did notexceed 4%. The spontaneous ⁵¹ Cr release for ST-13 type A synoviocyteswas 38%.

FIG. 17. Assessment of patient benefit. There are several standardcriteria to assess whether arthritis patients are benefiting fromtreatment. At each visit, patients are scored according to the tencriteria listed and an overall additive score is determined. This scoreis used to establish the effectiveness of the drug treatment. Otherparameters can also be measured. These include blood counts, liverfunction tests, Westegren sedimentation rates, rheumatoid factor tests,etc.

FIG. 18. Analysis of Vβ Gene Usage in Synovial Tissue Derived T CellLine. Line ST-2, derived from the synovial membrane infiltrating cellsof a rheumatoid arthritis patient, was analyzed for TCR Vβ expressionusing the cDNA, PCR amplification, slot blot hybridization protocol. Theleft part of the figure represents the autoradiograph obtained when thepanel of Vβ genes was hybridized with the ST-2 amplified TCR specificcDNA probe. The right part of the figure is the densitometry trace ofthe autoradiograph.

FIG. 19. Detection of Vβ Gene Usage in Rheumatoid Arthritis T Cells.This figure is a tabulation of the results of the expression of thepanel of Vβ genes in 12 paired synovium tissue derived and peripheralblood derived T cell lines from rheumatoid arthritis patients. For thetop part of the figure, the vertical axis represents the number ofsamples that were positive for a particular Vβ. The individual Vβ genesare indicated on the horizontal axis. Data derived from synovial T cellsand peripheral blood T cells are plotted in pairs as the open andcrosshatched bars, respectively. For the bottom part of the figure, thefrequencies of the individual Vβ genes in the 12 patient samples areshown (% synovial and % PBT). To indicate preferential usage of Vβ genesthe ratio of occurrence in the synovial/peripheral blood samples isshown.

FIG. 20. Detection of Dominant Vβ Gene Usage in Rheumatoid Arthritis TCells. This figure is similar to FIG. 19, except that the tabulated dataincludes only the expression of the most frequently occurring Vβ genesas determined by the densitometry trace. The most frequent or dominantVβ was determined from the highest peak height which was used as astandard. Any Vβ gene with a corresponding densitometry peak with heightgreater than 50% of the standard was used in the tabulation.

FIG. 21. Detection of Vα Gene Usage in Rheumatoid Arthritis T Cells.This figure is similar to FIG. 19, except the patient samples wereanalyzed for Vα gene expression. The data in this figure represents thetotal Vα expression observed, not the dominant or most frequentlyexpressed Vα. 85% of the T cell samples tested expressed Vα10 at adensitometry peak height 100-fold greater than for the other Vα genes.

FIG. 22. Vα Gene Usage in the Synovial Tissue Derived T Cell Line ST-5.The dominantly expressed Vα gene is Vα10, although other Vα genes areexpressed as minor populations.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to monoclonal antibodies whichrecognize defined regions of T cell antigen receptor (TCR) chains. Suchantibodies bind to epitopes of the variable, diversity, joining, and/orconstant regions of the alpha, beta, gamma, or delta chains of the Tcell antigen receptor. As used herein, a monoclonal antibody reactivewith the "variable region" of the TCR shall be construed to be amonoclonal antibody reactive with an epitope of the V region or acombination epitope of the V region or a combination epitope of the V-Dor V-D-J regions; a monoclonal antibody reactive with a "variableregion" of TCR may recognize an idiotype, a clonotype, or, preferably,may recognize a minor framework region expressed by a subgroup of Tlymphocytes. The term minor framework region refers to a region of theTCR which is not shared by all TCR molecules, but is also not unique toa particular clone of T cells. In a specific embodiment, the monoclonalantibodies of the invention are reactive with a constant region of thealpha chain of the T cell antigen receptor. In another embodiment, themonoclonal antibodies are reactive with a variable region of the betachain of the T cell antigen receptor. In particular, such an anti-TCRβmAb can recognize Vβ5.3. In another particular embodiment, such ananti-TCRβ mAb can recognize Vβ8.1. In another embodiment, the monoclonalantibodies are reactive with a variable region of the delta chain of theT cell antigen receptor. In particular such an anti-TCRδ mAb canrecognize Vδ1.

The monoclonal antibodies of the invention, and fragments, derivatives,and analogues thereof, have uses in diagnosis and therapy.

In specific embodiments of the invention, monoclonal antibodies whichbind to Vδ1, Vβ3, Vβ9, or Vβ10 variable regions of the T cell antigenreceptor may be utilized in the diagnosis and therapy of rheumatoidarthritis.

5.1. GENERATION OF MONOCLONAL ANTIBODIES REACTIVE WITH DEFINED REGIONSOF THE T CELL ANTIGEN RECEPTOR

The monoclonal antibodies of the invention are directed to an epitope ofa defined region of the T cell antigen receptor. A monoclonal antibodyto an epitope of the T cell can be prepared by using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include but are not limited to the hybridomatechnique originally described by Kohler and Milstein (1975, Nature256:495-497), and the more recent human B cell hybridoma technique(Kozbor et al., 1983, Immunology Today 4:72), EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96), and trioma techniques.

In one embodiment, the monoclonal antibodies may be of human monoclonalantibodies or chimeric human-mouse (or other species) monoclonalantibodies. Human monoclonal antibodies may be made by any of numeroustechniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:7279;Olsson et al., 1982, Meth. Enzymol. 92:3-16). Chimeric antibodymolecules may be prepared containing a mouse (or rat, or other species)antigen-binding domain with human constant regions (Morrison et al.,1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851; Takeda et al., 1985, Nature314:452). For therapeutic purposes, antibodies can be further humanized,by producng human constant region chimeras, where the conserved orframework regions of the antigen-binding domain are also from humansequences, and only the hypervariable regions are non-human.

As part of the production of the monoclonal antibodies of the invention,various host animals, including but not limited to rabbits, mice, andrats can be immunized by injection with purified T cell antigenreceptors or polypeptides or fragments thereof, a recombinant orsynthetic version thereof, or T lymphocytes.

Various cell lines can be used as immunogens to generate monoclonalantibodies to the V region of human T cell antigen receptors, including,but not limited to, those cell lines listed in Table 4 (Toyonaga et al.,1987, Ann. Rev. Immunol. 5: 585-620). Any T cell line could be used,including any γ δ + cell line, such as (but not limited to) PEER, MOLT13, WM 14, AK119, etc., provided that TCR is expressed on the cellsurface, as in the MOLT 13 cell line. Note that antibodies to known V,D, J, DJ, VJ, VDJ or combinations thereof can also be generated byimmunizing with these cell lines. V, D, J, and C region α, β, γ or δexpression can be determined in any cell line by well known proceduresincluding cDNA sequencing, in situ hybridization, polymerase chainreaction analysis, Northern analysis, Southern analysis, immunoassay, orflow cytometry, to name but a few.

Whole cells that can be used as immunogens to produce TCR specificantibodies also include recombinant transfectants. For example, β⁻Jurkat cells can be reconstituted by transfection with an β cDNA toproduce intact αβ TCR on the cell surface (Ohashi, P. S., et al.,Nature, 316:606-609). These transfectant cells could then be used asimmunogen to produce α or β TCR antibodies of preselected specificity.Other examples of such transfectant cells have been reported (Kaye, J.and Hedrick, S. H., 1988, Nature 336:580-583; Dembic, Z., et al., 1986,Nature 320: 232-238; Saito, T., et al., 1987, Nature 325:125-130), butany procedure that works to express transfected TCR genes on the cellsurface could be used to produce the whole cell immunogen. Thisincludes, but is not limited to, eukaryotic expression systems utilizinga phospholipid anchor domain (International Patent Application no.PCT/US88/02648 published Feb. 9, 1989).

Screening procedures that can be used to screen hybridoma cellsexpressing different anti-TCR antibodies include but are not limited to(1) enzyme linked immunosorbent assays (ELISA), (2) flow cytometry(FLOW) analysis, (3) immunoprecipitation, and (4) the ability tocomodulate the CD3 antigen (part of the TCR-CD3 complex present on thesurface of T cells) off of the surface of cells. The comodulation andFLOW screening procedures are preferred for the selection of antibodiespotentially useful in therapy since these procedures will result inselection of antibodies that are able to recognize intact TCR on livecells due to the inherent properties of these techniques.

Many different formats of an ELISA that can be used to screen foranti-TCR antibodies can be envisioned by one skilled in the art. Theseinclude, but are not limited to, formats comprising purified,synthesized or recombinantly expressed TCR polypeptide attached to thesolid phase or bound by antibodies attached to the solid phase orformats comprising the use of whole T cells or T cell lysate membranepreparations either attached to the solid phase or bound by antibodiesattached to the solid phase. Samples of hybridoma supernatants would bereacted with either of these two formats, followed by incubation with,for instance, a goat anti-mouse immunoglobulin complexed to anenzyme-substrate that can be visually identified.

In another screening method, supernatants of antibody producinghybridomas can be screened by FLOW in a number of different ways as isknown by one skilled in the art. One screening procedure involves thebinding of potential antibodies to a panel of T cells that expresswell-known TCRs on their surface. Generally, antibodies that react withintact TCR are detected by this analysis, but the T cells can be fixedslightly with e.g. ethanol, in which case antibodies reacting withdenatured TCR polypeptide can be identified. FLOW assays can also beformatted where potential antibodies are screened by their ability tocompete with the binding of a known antibody for the TCR present on a Tcell.

It is also possible to screen antibodies by their ability toimmunoprecipitate a known TCR as analyzed by SDS-polyacrylamide gelelectrophoresis or Western blot analysis. An advantage to this assay isthat it is possible to identify the chain of the TCR heterodimer thatthe anti-TCR antibodies are recognizing.

Additionally, screening may be carried out by observing the comodulationof the CD3 antigen. The TCR normally exists on the surface of T cells asa complex with the chains of the CD3 complex. When an antibody binds tothis TCR:CD3 complex, the complex in turn becomes internalized in the Tcell and disappears from the cell surface. Thus, if T cells are reactedwith an antibody specific to the TCR and the complex becomesinternalized, a further reaction with an anti-CD3 specific antibody willresult in substantially no detection of CD3 bearing cells by FLOWanalysis. This comodulation screening procedure detects antibodies thatare able to interact with intact TCR on the surface of T cells.

Many additional screening assays, such as those based upon competitionwith anti-TCR antibodies of known specificity or the ability to cause Tcells expressing known TCRs to proliferate in culture, will be known tothose skilled in the art and can be used for the selection ofappropriate antibodies.

A molecular clone containing a DNA sequence of an antibody to an epitopeof a T cell surface molecule can be prepared by known techniques.Recombinant DNA methodology (see e.g., Maniatis et al., 1982, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.) may be used to construct nucleic acid sequences whichencode a monoclonal antibody molecule, or antigen binding regionthereof.

Antibody molecules may be purified by known techniques, e.g.,immunoabsorption or immunoaffinity chromatography, chromatographicmethods such as HPLC (high performance liquid chromatography), or acombination thereof, etc.

Once antibodies of the desired specificity are generated, otherantibodies of the same epitope specificity can be selected. Antibodiesof such similar epitope specificity can be selected, for example, byobserving the ability of such antibody or binding region thereof toinhibit the binding of the antibody of known specificity to its antigen.Various competitive binding assays known in the art can be used.

5.2. FRAGMENTS AND DERIVATIVES OF MONOCLONAL ANTIBODIES REACTIVE WITHDEFINED REGIONS OF THE T CELL ANTIGEN RECEPTOR

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab')₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab' fragments which can begenerated by reducing the disulfide bridges of the F(ab')₂ fragment, andthe Fab fragments which can be generated by treating the antibodymolecule with papain and a reducing agent.

Various chemical derivatives of the antibodies of the invention can alsobe produced. For example, immunoconjugates consisting of an antibodymolecule or binding region thereof bound to a label such as aradioisotope or other tracer molecule can be made by techniques known inthe art. Alternatively, the antibody molecule or fragment thereof can bebound to a therapeutically useful molecule which is targeted to itsdesired site of action by virtue of the antibody's binding specificity.As one example of such an embodiment, a cytotoxic compound can beconjugated to an antibody of the invention which is specific to a regionof the TCR on lymphocytes which are the causative agents of anautoimmune disorder. The cytotoxic compound in conjugate form is thustargeted to the implicated T lymphocytes.

In addition, isotypes of the antibodies of the invention can be switchedin order to optimize clinical applications. For example, some isotypes(such as IgG2a) are superior effectors of antibody dependent cellcytotoxicity reactions. Likewise, some isotypes (such as IgG2a) are morereadily eliminated from the circulation via the Fc receptors present oncells of the reticuloendothelial system and sequestered in the spleenthan others, and, if bound to a target cell (which, for example, may bean effector of autoimmune disease), would be more efficient at removingthe target cell from sites of active disease. Accordingly, certainantibody isotypes may be preferable to others, depending on the intendedclinical application. Therefore, hybridomas are screened for TCRspecific mAbs using a TCR ELISA designed to select for desirableisotypes, such as IgG. In addition, a different isotype may be generatedby spontaneous isotype switching and directed toward various uses. Amethod exists which facilitates selecting for the isotype of interest;the procedure for isotype switching of IgG1 to IgG2a presented as anexample, is as follows:

Hybridoma cells are grown in log phase for a 2-3 week period prior to amagnetic bead negative selection. For the magnetic bead selection, superparamagnetic iron oxide particles coated with a goat anti-mouse antibodypreparation including all IgG isotype classes (Biomag® beads purchasedfrom Advanced Magnetics, Inc.) may be used. For the process of switchingan antibody isotype from IgG1 to IgG2a, it is necessary to block theIgG2a binding sites on the antibody coated beads by incubating with anirrelevant antibody having an IgG2a isotype. 10⁸ hybridoma cells arethen incubated with the beads, allowing cells expressing IgG1, IgG2b andIgG3 isotypes to bind to the beads and be removed magnetically from thepopulation. This negative selection should be repeated several times.The remaining cell population, depleted of IgG1, IgG2b and IgG3 bearingcells and enriched for IgG2a bearing cells is plated in 15 microtiterplates at a cell density of about 1000 cells per well. Usingcommercially available anti-isotypic reagents in an ELISA assay, thewells are screened for IgG2a production. Wells should be screened againby ELISA for IgG2a production and positive clones replated at 0.3 cellsper well followed by another round of screening and replating at 0.3cells per well. Approximately 1-5 out of 10⁷ cells which have switchedisotype are optimally selected. Switches such as IgM to IgG can be donesimilarly using the appropriately coated antibody-coated beads.

Accordingly, the isotype of TCSδ1 also referred to as δTCAR-3, describedin U. S. patent application Ser. No. 115,256 filed Oct. 29, 1987,produced by hybridoma δTCAR-3, deposited with the ATCC and assignedaccession number HB9578, was switched from IgG₁ to IgG_(2a) by the abovemethod.

5.3. IMMUNOASSAYS

The antibodies of the invention, and fragments thereof containing thebinding region (e.g., Fab, Fab', F(ab')₂), can be used in variousimmunoassays. Such immunoassays include but are not limited tocompetitive and non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"immunoassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays, and immunoelectrophoresis assays, to name but a few.

5.4. DIAGNOSIS AND THERAPY

The antibodies of the invention, and fragments and derivatives thereof,can be valuable in the diagnosis and therapy of various conditions anddisorders affecting the immune system.

5.4.1. DIAGNOSIS

In various embodiments, the antibodies, derivatives and fragmentsthereof of the invention can be used to detect, quantitate, and/orlocalize T lymphocytes which express TCRs that comprise and expose thedefined region to which the antibody can bind. Both in vitro and in vivoassays can be used, including but not limited to the assays described inSection 5.3, supra. In addition, imaging techniques can be used, inwhich an antibody of the invention or derivative or fragment thereof isbound to a label. The labeled antibody can then be administered in vivoto determine the localization of its target antigen.

In particular embodiments of the invention, a lymphatic malignancy orimmune disorder may be diagnosed by detecting the immunospecific bindingof a monoclonal antibody, or derivative or fragment thereof, reactivewith an epitope of a defined constant or variable region of a T cellantigen receptor in a patient sample. The patient sample may consist ofany body fluid, including but not limited to peripheral blood, plasma,cerebrospinal fluid, lymphatic fluid, peritoneal fluid, or pleuralfluid, to name but a few, or any body tissue. Binding may beaccomplished and/or detected in vitro or in vivo. In vitro binding maybe performed using histologic specimens or subfractions of tissue orfluid, i.e. substantially purified T cells. In vivo binding may beachieved by administering the antibody or fragment or derivative by anymeans known in the art (including but not limited to intravenous,intraperitoneal, intranasal, and intrasarterial, to name but a few) suchthat immunospecific binding may be detected; for example, by attaching aradioactive label to the diagnostic antibody, fragment, or derivative.

In specific embodiments of the invention, rheumatoid arthritis orFelty's syndrome may be diagnosed by detecting the binding of amonoclonal antibody, or fragment or derivative thereof, reactive with anepitope of Vδ1 (RA, FS), Vβ3, Vβ9, or Vβ10 (RA) variable region of a Tcell antigen receptor in a patient sample, such that increased bindingis detected in patients with rheumatoid arthritis or Felty's syndromecompared to normal controls.

In a preferred embodiment of the invention, rheumatoid arthritis orFelty's syndrome may be diagnosed by detecting binding of monoclonalantibody δTCS1, or fragments or derivatives thereof, in that theperipheral blood level of δTCS1 positive T cells in a patient withrheumatoid arthritis or Felty's syndrome is elevated compared to thelevel in the peripheral blood of a normal person. In a relatedembodiment, the percentage of total peripheral blood T cells which areδTCS1 positive is observed to be higher in a patient with rheumatoidarthritis or Felty's syndrome compared to the percentage of totalperipheral blood T cells which are δTCS1 positive in a normal person. Inadditional embodiments of the invention, a diagnosis of rheumatoidarthritis or Felty's syndrome may be made based on detecting elevatednumbers of δTCS1 positive T cells, relative to total T cells, in otherbody fluids and tissues including, but not limited to, synovial fluidand synovial membrane. In a further embodiment of the invention,rheumatoid arthritis or Felty's syndrome may be diagnosed in patients inwhich the peripheral blood ratio of δTCS1 positive to TCRδ1 positive Tcells is elevated and, preferably, that it is greater than about 0.4.The binding of antibody or fragments or derivatives thereof may bedetected in vitro or in vivo, as discussed supra. Intraarticularadministration of a labelled antibody or derivative or fragments thereofmay also potentially be utilized as a diagnostic procedure.

It should be understood that the diagnostic methods of the invention arebest used in the context of other diagnostic parameters in order toobtain a comprehensive patient diagnosis. For example, a diagnosis ofrheumatoid arthritis may be made based on the methods of the inventionin the context of other clinical features of rheumatoid arthritis, suchas typical joint involvement (chronic, symmetrical arthritis; earlyinvolvement most often in the hands); characteristic radiographicfeatures; the presence of rheumatoid factor; the presence of rheumatoidnodules, etc. (Fishman et al., Medicine, Second Edition, J. B.Lippincott Company, Philadelphia, pp. 340-346). As with any diagnosticcriteria, the parameters disclosed in the present invention may not besole determinants, or pathognomonic, of a particular disorder.

Alternatively, according to the invention, a lymphatic malignancy orimmune disorder may be diagnosed by detecting the presence of nucleicacid sequences homologous to a gene encoding a defined constant orvariable region of a T cell antigen receptor in mRNA from a patientsample. Several procedures could be used to correlate TCR geneexpression with disease. These involve 1) producing and analyzing cDNAlibraries obtained from the disease related T cells to determine thepresence of frequently used or dominant TCR genes. 2) Analyzing diseasesamples by Southern blot to determine whether specific geneticpolymorphisms (restriction fragment length polymorphisms, RFLPs) oroligoclonal TCR rearrangements exist. 3) Analyzing disease samples bythe cDNA synthesis, polymerase chain reaction amplification, and slotblot hybridization procedure, see Section 11, infra. The third procedurerepresents a more efficient procedure in the time required for analysisand in the number of patients that can be analyzed to detect a diseasecorrelation. A fourth procedure using in situ hybridization of T cellswithout prior T cell culturing may also be extremely useful. Once thedisease correlations of interest have been identified, then specific TCRbased therapeutics, e.g. anti-TCR monoclonal antibodies, may be produced(see 11.3 infra).

In specific embodiments of the invention, rheumatoid arthritis may bediagnosed in a patient by detecting the presence of nucleic acidsequences homologous to a gene encoding Vδ1, Vβ3, Vβ9, or Vβ10 variableregion of a T cell antigen receptor in mRNA from a patient sample, andfinding that more frequently expressed Vβ genes include Vβ3, Vβ9, andVβ10 and/or that a more frequently expressed Vδ gene is Vδ1.

5.4.2. THERAPY

In other embodiments of the invention, antibodies, derivatives, orfragments thereof directed against a defined region of the TCR can betherapeutically administered. For example, if the antibody is capable ofinducing in vitro T cell proliferation, it may be administeredtherapeutically to stimulate specific cell-mediated immunity. In anotherparticular embodiment, an antibody directed against a defined region ofthe TCR can be used to target a cytotoxic molecule to specific TCRswhich are the causative agents of an autoimmune disorder.

In another embodiment, an antibody may be administered therapeuticallyto block the interaction of effector T cells with their specific antigenand thus modulate a deleterious response. A further therapeuticembodiment is to administer an antibody therapeutically to bind to itstarget and mark those cells for elimination by the RES septem or byantibody dependent cell cytotoxicity (ADCC) reactions, the ablation ofthe target T cells resulting in a therapeutic effect. According tovarious embodiments of the invention, monoclonal antibodies directedtoward defined regions of T cell antigen receptors may either be usedtherapeutically at low mitogenic concentrations to specifically activatethe TCR bearing subset of interest, or alternatively could be used atmuch higher concentrations to bind to the receptors of interest, andthereby tag that T cell subset for elimination by the reticuloendotelialsystem. The important criteria for disease treatment is the ability tomodulate specific disease related T cell subsets. The exact nature ofthis therapeutic modulation, whether to block or suppress a particular Tcell subset, or alternatively, whether to stimulate and activate aparticular subset, will depend upon the disease of interest and thespecific T cell subset(s) involved.

According to various embodiments of the invention, the number of γδ+ Tcells with cytotoxic activity may be increased by exposing T cells to aneffective concentration of a monoclonal antibody, or derivative orfragment thereof, reactive with an epitope of the variable region of thedelta chain of the T cell antigen receptor. Alternatively, higherconcentrations of the same antibody may be effective in depleting γδ+cells. The concentrations of antibody which induce proliferation, orwhich result in depletion, of γδ+ T cells may be accomplished by methodsknown in the art, as exemplified in Section 9 below. In specificembodiments of the invention, δTCS1 monoclonal antibody, or fragments orderivatives thereof, used at different concentrations, may result inproliferation, increased cytotoxic activity, or decreased cytotoxicactivity, respectively, of T cell populations derived from patients withrheumatoid arthritis.

First generation treatments based on T cell receptor therapeutics may bedeveloped based upon the correlation of specific TCR Variable regionsubfamilies with disease. These treatments offer an improvement overcurrent procedures, such as anti-CD3 antibody treatment in patientsundergoing renal transplant rejection, as the whole T cell populationwill not be modulated; only the particular T cell subset expressing theTCR V region subfamily of interest will be modulated. In addition, TCRvariable region subfamily derived reagents are applicable to thetreatment of groups of patients showing similar expression; whereas, atherapeutic combination based on a specific V.sub.α (V-J-C) or aspecific V.sub.β (V-D-J-C) may only be useful for the treatment ofindividual patients expressing that exact TCR combination.

Second generation TCR based therapeutics, may include furtherrefinements of the association of particular variable (V), diversity (D)and joining (J) regions with specific disease states for α, β, γ and δTCR genes. Patients may be further subdivided into groups for treatmentbased upon the TCR V, D, J and C regions involved; with the goal beingto only modulate the actual disease related T cells and not to effectother T cell subsets.

In a particular embodiment of the invention, rheumatoid arthritis may betreated by administering therapeutically effective amounts of amonoclonal antibody, fragment or derivative thereof, which recognizes anepitope of the variable region of the beta chain of a T cell antigenreceptor. In particular embodiments, the variable region comprises Vβ3,Vβ9, or Vβ10.

In additional embodiments of the invention, a lymphatic malignancy orimmune disorder may be treated by administering therapeuticallyeffective amounts of a monoclonal antibody, or fragment or derivativethereof, which recognizes the constant region of the alpha chain or thevariable region of the delta chain of a T cell antigen receptor. In oneembodiment of the invention, rheumatoid arthritis or Felty's syndromemay be treated by administering therapeutically effective amounts of amonoclonal antibody, derivative, or fragment thereof, which recognizesan epitope of the Vδ1 variable region of the delta chain of a T cellantigen receptor. In a preferred embodiment, this monoclonal antibodyhas the binding characteristics of δTCS1. In a specific embodiment ofthe invention, a patient with rheumatoid arthritis may be treated byadministering milligram quantities per day of δTCS1 antibody, or afragment or derivative thereof, in a suitable pharmaceutical carrierinto a patient in need of such treatment (See Section 10, infra).

5.4.3. THERAPEUTIC COMPOSITIONS; KITS

Various delivery systems are known and can be used for therapeuticdelivery of the antibodies of the invention and fragments andderivatives thereof. Methods of introduction include but are not limitedto intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, and intranasal routes. In one embodiment, the antibody canbe encapsulated in liposomes.

Various pharmacologic compositions may be utilized in order to deliverthe antibodies, or fragments or derivatives thereof, according to theinvention. Any suitable pharmaceutical agent with desirable solubilitycharacteristics and chemical properties may be used, including but notlimited to, where appropriate, saline or dextrose solutions.

Kits for practice of the instant invention are also provided. Forexample, such a diagnostic kit comprises in one or more containers amonoclonal antibody or derivative or fragment thereof reactive with anepitope of the constant region of the α TCR. In other embodiments, theantibody or derivative or fragment is reactive with Vβ5.3, or Vβ8.1 ofthe TCR. The antibody may be labeled. Alternatively, the kit can furthercomprise a labeled binding partner of the antibody, derivative orfragment. Therapeutic kits can comprise the therapeutic compositions ofthe invention in one or more containers.

5.5. MONOCLONAL ANTIBODIES REACTIVE WITH THE CONSTANT REGIONS OF THE TCELL ANTIGEN RECEPTOR ALPHA POLYPEPTIDE

In a specific embodiment, the present invention relates to monoclonalantibodies reactive with the constant region of the alpha chain of theTCR. In a particular example, the invention relates to mAb αF1. Inanother particular embodiment, the invention relates to mAb αF2.

αF1 and αF2 recognize two different epitopes of the framework region ofthe constant region of the TCRα monomer. In various embodiments of theinvention, αF1 or αF2, or both, or fragments or derivatives of either,or both, can be used to bind to TCRα peptide amino acid sequences,either as part of an intact TCR complex or a peptide, or a fragmentthereof.

Monoclonal antibodies αF1 and αF2 are described more fully in thesubsections below and in Section 6, infra.

5.5.1. BINDING SPECIFICITIES OF αF1 AND αF2

Monoclonal antibodies αF1 and αF2 were found to react with αβ TCR⁺ Tcell lines, but did not react with αβ TCR⁻ T cell lines (includingγδTCR⁺ cell lines) or B cell lines. Since αF1 and αF2 react with TCR onmany T cell lines and a large percentage of PBLs, they are reactive withmajor framework regions of the TCR molecule.

Both mAb αF1 and αF2 immunoprecipitate the 90 kD αβ TCR heterodimer. mAbαF1 reacts with the oligopeptide representing amino acid residues141-159 of the α peptide, whereas αF2 reacts with the oligopeptiderepresenting amino acid residues 212-231. Both oligopeptides residewithin the constant region of the α chain sequence. Both αF1 and αF2selectively immunoprecipitated in vitro synthesized peptide.

5.5.2. IMMUNOHISTOLOGICAL ANALYSIS USING αF1 AND αF2

Although both αF1 and αF2 immunoprecipitate TCR of cell lysates ofdiverse T cell lines, neither bind to viable T cells. Presumably this isbecause the epitopes recognized by αF1 and αF2, on the constant regionof α chain TCR, are hidden on the cell surface.

When the integrity of the cell membrane is disrupted, for example, byhistologic fixative, αF1 and αF2 can be used to bind to TCR α peptide.By immunohistological staining, both αF1 and αF2 recognize greater than90% of both mature peripheral resting and activated T cells,corresponding to the distribution of αβ TCR in the adult T cellpopulation.

5.5.3. USES OF αF1 AND αF2

Because αF1 and αF2 bind to epitopes within the constant region of TCR αpeptide, both mAb can be used to define populations of TCR α⁺ cells.

For example, in one embodiment of the invention αF1 and αF2 can be usedto study TCRα expression during thymocyte differentiation. It is thoughtthat thymocytes differentiate and mature gradually as they pass fromthymic cortex to medulla and finally to peripheral blood (Benjamini andLeskowitz, 1988, Immunology A Short Course, Alan R. Liss, Inc., New Yorkp. 141). Numerous studies have suggested that in thymus, during T celldifferentiation, δ, β, and γ chain genes are rearranged and transcribedearlier than α chain genes (Chien et al., 1984, Nature 312:31; Raulet etal., 1985, Nature 314:103; Snodgrass et al., 1985, Nature 315:332). αF1and αF2 can be used to correlate the expression of TCR α protein withthymocyte development.

Furthermore, αF1 and αF2 can be used to determine the state of αTCRprotein expression in a cell. For example, unstimulated CEM cellsexpress neither TCR nor CD3 on their surface; however, stimulation withPMA induces CD3 expression (Schackelford et al., 1987, J. Immunol,138:613). Using αF1, the unstimulated CEM line was found to be negativefor the expression of α chain TCR protein in the cytoplasm; however,stimulation with PMA resulted in an increase in intracellular TCRα andsurface expression.

αF1 and/or αF2 can also be used to generate or select monoclonalantibodies directed at unique epitopes of the TCRα constant region, i.e.by competitive binding studies. Novel mAb reacting with differentepitopes of the TCR α constant region would not compete with αF1 or αF2for TCR α binding. αF1 and αF2 can also be used to generate antibodiesto the αTCR constant region that are able to react with viable cells.

αF1 and αF2 can also be valuable in diagnosis and therapy, as describedinfra.

In one embodiment of the present invention, αF1 and/or αF2 can be usedto detect TCR α protein expression in specimens in vitro, e.g., in celllysates or in histologically prepared specimens. In one particularembodiment, classification of lymphatic malignancies into B cell, Tcell, or non-B non-T cell groups, can be greatly facilitated bydemonstrating the presence or absence of TCR a protein using αF1 and/orαF2 according to the present invention.

αF1 and αF2, in various embodiments of the present invention, can beused to establish the cellular derivation of lymphatic malignancies,such as leukemia, lymphoma, and myeloproliferative diseases, and mayprove useful in the diagnosis of other malignancies or non-malignantdisorders of the immune system.

In another embodiment of the present invention, αF1 and αF2 can be usedto monitor therapies involving the proliferation or ablation of α⁺ Tcells.

In a further embodiment of the present invention, αF1 and αF2 could beused to detect TCR-α-producing tissues in vivo. Because αF1 and αF2 donot bind to viable cells, binding would only occur to cells whichexhibit enhanced permeability, such as moribund or necrotic cells. Thesecells would, most likely, be localized to malignant tumors or similarneoplasms associated with lymphoid cell death. In an embodiment of theinvention, αF1 and αF2 could be conjugated to a label comprising, forexample, a radioisotope. Labeled αF1 and αF2 could then be injected intoa patient; localization of radiolabeled antibody can reveal tumor sites.

5.6. MONOCLONAL ANTIBODIES REACTIVE WITH THE VARIABLE REGION OF THE TCELL ANTIGEN BETA POLYPEPTIDE

In another specific embodiment, the present invention relates tomonoclonal antibodies reactive with specific variable regions of thebeta chain of the TCR. In one particular embodiment of this aspect ofthe invention, the invention relates to a monoclonal antibody reactivewith Vβ5.3. An example of such a Vβ5.3-specific antibody is mAb W112. Inanother particular embodiment, the invention relates to a monoclonalantibody reactive with Vβ8.1. An example of such a mAb, reactive withhuman Vβ8.1, is mAb 2D1.

In various embodiments of the invention, W112 or 2D1, or fragments orderivatives of either, can be used to bind with TCRβ peptide amino acidsequences, either as part of an intact TCR complex or β peptide, or Tcell surface protein, or a fragment thereof.

Monoclonal antibodies W112 and 2D1 are described more fully in thesubsections below and in Section 7, infra.

5.6.1. BINDING SPECIFICITIES OF W112 AND 2D1

W112 immunoprecipitates a heterodimer of 48 kd and 40 kd from HPB, and2D1 immunoprecipitates the same size dimer from Jurkat cell lysates.Both W112 and 2D1 are anti-minor framework antibodies as they react witha minority of peripheral blood T cells, indicating specificity directedtoward the variable portion of TCRβ. Western blot analysis showed thatboth W112 and 2D1 reacted with the TCRβ protein of their respective celllines. W112 is a TCR Vβ5.3 specific reagent and 2D1 is a TCR Vβ8.1specific reagent.

5.6.2. USES OF W112 AND 2D1

Because both W112 and 2D1 are anti-minor framework TCRβ specificreagents , these mAb can be used to study, define, quantitate, andlocalize a subset of T cells.

In one embodiment of the present invention, W112 and 2D1 can be used tostudy Vβ5.3 and Vβ8.1 expression during thymocyte differentiation. Inanother embodiment, W112 and 2D1 can be used to generate or selectadditional anti-minor framework antibodies, i.e., by competitive bindingstudies.

W112 and 2D1 can also be valuable in diagnosis and therapy, as describedinfra.

In one embodiment, W112 and/or 2D1 can be used to detect Vβ5.3 and Vβ8.1subsets of T cells in specimens in vitro, e.g., in cell lysates or inhistologically prepared specimens. Such use is of value in the diagnosisof patients with immune system disorders affecting such subsets of theirT lymphocytes. In an embodiment of the present invention, W112 and 2D1can be used to monitor therapeutic procedures that effect these subsetsof T cells.

Since both W112 and 2D1 react with viable cells, they can be usedtherapeutically to modulate Vβ5.3 and Vβ8.1 expressing T cells involvedin disease. In one embodiment, W112 or 2D1 could be administeredtherapeutically to ablate the Vβ5.3 or Vβ8.1 T cell subsets. In anotherembodiment, W112 or 2D1 could be administered therapeutically to inducethe proliferation of the Vβ5.3 or Vβ8.1 T cell subsets. In theseembodiments the selective ablation or proliferation of a specific T cellsubset by anti-minor framework antibodies will be preferred tomodulating the whole T cell population with anti-major framework orpan-T reagents, as only the disease specific T cell subsets will beeffected.

In another embodiment of the present invention, W112 or 2D1 could beconjugated to a label comprising, for example, a radioisotope. LabeledW112 or 2D1 could then be injected into a patient; localization ofradiolabeled antibody can reveal Vβ5.3 or Vβ8.1 specific disease sites.

6. EXAMPLE THE GENERATION OF TWO MONOCLONAL ANTIBODIES αF1 AND αF2,DIRECTED AGAINST TWO DIFFERENT EPITOPES OF THE HUMAN αTCR CONSTANTREGION

We describe herein two mAb, termed αF1 and αF2, generated againstpurified αβ heterodimer TCR. Using synthetic oligopeptides, wedemonstrated that αF1 recognized amino acid residues 141-159, and αF2recognized amino acids 212-231, of the constant region of the α chainTCR. Although neither mAb reacted with viable T cells, both antibodiesimmunoprecipitated TCR αβ heterodimer from HPB, Jurkat, and PBL cells, αchain protein of PMA-stimulated CEM cell line, and a 32 kD in vitrotranslation product of α chain cDNA. These antibodies have proved to beuseful in the immunohistochemical staining of human tissues. These twomAb are valuable tools in the study of TCR and in the clinicalclassification of T cell lineage neoplasms.

6.1. MATERIALS AND METHODS

6.1.1. CELL LINES

The human cell lines HPB-ALL, Jurkat, Daudi, U923, CEM and Molt-4 wereobtained from the American Type Culture Collection (Rockville, Md.), andwere maintained in RPMI-1640 medium supplemented with 10% fetal bovineserum. Sequence analyses of the HPB-ALL and HPB-MLT has indicated thatboth cell lines have the same β and α TCR genes and may have beenconfused during in vitro culturing (Berkhout et al., 1988, Nucleic AcidsResearch 16:5209). These cells are referred to as HPB cells to indicatethat they are the same line. The cell line Molt-13 was supplied by Dr.Michael Brenner, Harvard Medical School, Boston, Mass. TIL21 andTIL21pBT are human T cell lines isolated from infiltrated lymphocytes oflung tumor (Flatow et al., 1988, FASEB J. 2:3505). Human peripheralblood lymphocytes (PBL) were isolated from normal donors usingFicoll-Hypaque gradient separation (Pharmacia, Piscataway, N.J.).

6.1.2. MONOCLONAL ANTIBODIES

The murine hybridomas 3A8 and 3D6 were generated by immunizing eightweek old female Balb/c mice (Jackson Laboratories, Bar Harbor, Me.) with1.0 μg of purified αβ TCR protein in complete Freund's adjuvantintraperitoneally. At two-week intervals the mice were given 1.0 μgpurified αβ TCR in incomplete Freund's adjuvant intraperitoneally. Afterthree months, sera were tested to be positive as determined byimmunoprecipitation of TCR heterodimer using ¹²⁵ I-labelled HPB andJurkat cell lysates. Four days before fusion, the mice received thefinal intravenous injection of 1 lg of purified TCR in PBS. Hybridomacells were generated using an established protocol (Wu et al., supra).The mAb produced by clones 3A8 and 3D6 were designated as αF1 and αF2,respectively. The isotypes of mAb αF1 and αF2 were determined to beIgG2a and IgG2b, respectively, using a commercial typing kit (Zymed, SanFrancisco, Calif.). βF1 (Brenner et al., 1987, J. Immunol. 138:1502),and δTCS-1 (Wu et al., supra.) (T Cell Sciences, Cambridge, Mass.) aremAb that react with the β and δ chains of TCR, respectively. OKT3 waspurchased from Ortho Diagnostic System (Raritan, N.J.)

6.1.3. T CELL LYSATES AND αβ TCR PROTEIN

Human T cell lines bearing the appropriate TCR were solubilized at 5×10⁷cells/ml in lysis buffer containing 10 mM Tris PH 8.0, 1% Nonidet -P 40(NP-40), 10 mM iodoacetamide (IAA), 1 mM phenylmethyl sulfonyl fluoride(PMSF), 0.04% aprotinin and 0.3 mM N-tosyl-L-phenylalanine chloromethylketone (TPCK). HPB cell lysates were applied to a mixed lectin column oflentil- and ricin-agarose column (Sigma, St. Louis, Mo.). The effluentfrom the lectin column was further purified through a column of mAb βF1immobilized Affi-gel 10 (Biorad, Rockville Centre, N.Y.). The bound αβTCR protein was then eluted with solution containing 25 mM ofdiethylamine, pH 11.5 and 0.2% NP-40. In general, approximately 80 μg ofαβ TCR protein were purified from 5×10¹⁰ HPB-ALL cells.

6.1.4. ELISA METHOD FOR SCREENING mAb (FIG. 6A)

96-well microtiter plates (Dynatech, Alexandria, Va.) were coatedovernight at 4° C. with 100 μl per well of goat anti-mouse IgGFc-specific antibodies (Cappel, West Chester, Pa.) at 1 μg/ml in PBS.This was designed to preferentially select for monoclonal antibodieshaving an IgG isotype. Plates were then blocked with 100 μl per well of1% BSA in PBS containing 0.05% Tween-20 for 30 minutes. 100 μl ofhybridoma supernatants were added to each well and incubated for 1 hour.100 μl of cell lysate containing 25% FCS and 0.1 μg/ml ofbiotin-labelled βF1 F(ab)₂ fragment were then added to each well andincubated for an additional 2 hours. After washes, 100 μl horseradishperoxidase (HRP) conjugated streptavidin (Zymed, San Francisco, Calif.)was added at 1:2500 dilution to each well. After 30 minutes ofincubation, 50 μg of the 0.1% O-phenylenediamine dihydrochloride (OPD)(Sigma, St. Louis, Mo.) substrate solution was added to each well.

The reactions were stopped with 100 μl of 2N H₂ SO₄. The absorbance ofeach well was then read on a microtiter plate reader (Dynatech,Cambridge, Mass.) at 490 nm. Between each step, ELISA plates were washedfour times with 250 μl per well of PBS containing 0.5% Tween-20 and theincubations were carried out at room temperature.

6.1.5. ASSAY FOR TCR PEPTIDE BINDING

TCR oligopeptides were custom synthesized by Pennisular Laboratory(Belmont, Calif.) according to the deduced sequences from the α-andβ-chain TCR genes (Yanagi et al., 1985, Proc. Nat;. Acad. Sci. U.S.A.82:3430; Yanagi et al., 1984 Nature 308:145-149). TCR oligopeptides werecoupled to protein carrier, BSA (Sigma, St. Louis, Mo.) withsuccinimidyl-4-(p-maleimidophenyl) butyrate (Pierce, Rockford, Ill.)according to the published procedure (Gitman et al., 1985, Proc. Natl.Acad. Sci. U.S.A. 82:7309). 100 μl of 4.0 μg/ml the peptide conjugateswere added per well to microtiter plates and incubated overnight at 4°C. Plates were blocked with PBS containing 1% BSA and 0.05% Tween-20 for30 minutes. 100 μl of each of the test mAb at about 10 μg/ml were addedto each well and incubated for 2 hours. After washing, 100 μl per wellof 1:1000 dilution of HRP-conjugated goat anti-mouse IgG antibodies(Zymed, San Francisco, Calif.) were added to each well of the plate andincubated for an additional two hours, followed by the addition of 0.1%of OPD substrate solution. The reactions were stopped with 100 μl of 2NH₂ SO₄. The absorbance of each well was then read on a microtiter platereader (Dynatech, Cambridge, Mass.) at 490 nm. Between each step, ELISAplates were washed four times with 250 μl per well of PBS containing0.5% Tween-20 and the incubations were carried out at room temperature.

6.1.6. IMMUNOPRECIPITATION AND SDS-PAGE.

Cells were harvested at log growth phase and their surface proteins werelabelled with ¹²⁵ I by the lactoperoxidase method (Brenner et al., 1984,J. Exp. Med. 160:541). Lysates of the iodinated T cells were prepared ina manner similar to that described above. Immunoprecipitation and onedimensional SDS-PAGE using 10% gels under either non-reducing orreducing conditions were performed as previously described (Wu et al.,supra).

6.1.7. IMMUNOHISTOLOGICAL TISSUE SECTION ANALYSIS

Culture cells were cytocentrifuged on glass slides (Cytospin, Shandon,Pittsburgh, Pa.) and fixed with 100% methanol. The immunoperoxidasestaining of cytospun cell smears was carried out using commercialstaining kits (CRL, Cambridge, Mass.). Immunoperoxidase staining wasalso carried out on frozen cryostat sections of thymus and tonsil.

6.1.8. IN VITRO TRANSLATION

Phage Sp6 RNA polymerase and rabbit reticulocyte in vitro translationsystem were purchased from New England Biolabs (Beverly, Mass.) andBethesda Research Laboratories (Gaithersburg, Md.), respectively.pGA5-pSP73 plasmid construct was a gift of Dr. Michael Brenner, HarvardMedical School, Boston, USA. pGA5-pSP73 is a plasmid construct composedof a pSP73 plasmid backbone (Promega, Wis., USA) and a coding region forthe α chain TCR gene derived from the pGA5 cDNA clone (Sim et al., 1986,Nature 312:771). pGA5-pSP73 plasmid construct was linearized withrestriction enzyme XhoI, and was subsequently used as the DNA templatefor the transcription of the pGA5 α chain gene using the phage Sp6promoter (Krieg et al., 1984, Nucl. Acids Res. 12:7057; Hope and Struhl,1985, Cell 43:177). The resulting capped pGA5 RNA transcript was thentranslated into ³⁵ S-labeled α chain protein in the rabbit reticulocytein vitro translation system according to the manufacturer's protocol.Rabbit globin mRNA was also included in the in vitro translation systemas an added irrelevant protein for the immunoprecipitation assays.

6.2. RESULTS

6.2.1. ISOLATION OF MAJOR FRAMEWORK mAb.

Both mAb αF1 and αF2 tested positive in the ELISA using cell lysates ofαβTCR⁺ HPB and Jurkat cell lines, but tested negative with cell lysatesof MOLT-4 and Daudi. MOLT-4 is a T cell line that lacks α chain TCR andDaudi is a B cell line that is αβ TCR negative. The mAbs were furtheranalyzed by immunoprecipitation using ¹²⁵ I-labeled HPB and MOLT 13 celllysates. Both mAb immunoprecipitated a 90 kD protein of TCR α and βheterodimer from HPB cells under non-reducing conditions (FIG. 1a, lanes1 and 2). The same αβ TCR protein is immunoprecipitated by βF1, a mAbdirected against β framework of TCR (FIG. 1A, lane 3) (Brenner et al.,1987, J. Immunol. 138:1502). Under reducing conditions, both αF1 and αF2mAb immunoprecipitated two proteins of 40 and 49 kD from HPB cells, asdoes βF1 (FIG. 1B, lanes 1, 2 and 3). In contrast, no proteins wereimmunoprecipitated by either αF1, αF2, or βF1 when membrane lysates fromMolt-13, a γδ T Cell line, was used (FIGS. 1A and 1B, lanes 5, 6, 7).

As a control, the Vδ specific antibody, δTCR-1, immunoprecipitated theγδTCR from the γδ⁺ cell line MOLT-13, but not from the αβ cell line HPB(FIGS. 1A and 1B, lanes 4 and 8).

Similar results were also obtained with cell lysates isolated from ¹²⁵I-labeled αβ⁺ Jurkat cells and PBLs (FIG. 2).

6.2.2. REACTIVITY OF mAb αF1 AND αF2

Immunocytochemical staining using αF1 and αF2 mAb was performed on avariety of cell lines. As shown in Table 1, both αF1 and αF2 mAb reactedpositively with αβ T cell lines HPB, Jurkat, TIL21 and TIL21PBT, butnegatively with γδ T Cell line Molt-13 and non-T cell lines Daudi andU923. Also αF1 and αF2 detected the presence of α chain TCR protein inthe PMA stimulated CEM line, but not in the unstimulated cells.

                  TABLE 1                                                         ______________________________________                                        CROSS REACTIVITY OF mAb WITH DIFFERENT HUMAN                                    CELL LINES                                                                                 Reactivity with mAb                                            Cell Name Cell Type                                                                              OKT3    BF1  αF1                                                                          αF2                                                                          δTCS1                         ______________________________________                                        HPB       T Cell   ++      +++  +++  +++  -                                     Jurkat T Cell ++ +++ +++ +++ -                                                TIL21 T Cell ++ ++ ++ ++ -                                                    TIL21pBT T Cell ++ ++ ++ ++ -                                                 CEM T Cell + ++ -- - -                                                        CEM+PMA**  ++ ++ ++ + -                                                       Molt-13 T Cell ++ - - - +                                                     Daudi B Cell -- -- -- -- --                                                   U923 Monocyte -- -- -- -- --                                                ______________________________________                                         *Cell growing at log phase were washed and cytospinned onto glass slides.     Immunohistological staining with different mAb was performed as described     Reactivity was determined arbitrarily on a scale of - to +++.                 **CEM cells were incubated with PMA at 1 ng/ml for three days before bein     harvested and stained with mAb.                                          

Both αF1 and βF1 were used to stain cryostat sections of human thymusand tonsil. As shown in FIG. 3, βF1 stained about 70% of T cells in thecortical zone (3A) and over 90% in the medullary zone (3C) of thethymus. αF1, however, stained about 30% T cells in cortex (3B) and 90%in medulla (3D). When tested on tonsil tissue section, both mAb reactedwith T cells in the interfollicular areas (E and F). αF2 staining wassimilar but weaker than that of αF1. Neither mAb stained viable T cellsat a detectable level.

6.2.3. SPECIFICITY OF αF1 AND αF2 mAb

To further define the specificity of αF1 and αF2, both mAb were testedfor their ability to recognize synthetic oligopeptides derived fromα-chain TCR sequence in an ELISA. The reactivities of αF1 and αF2against α and β chain derived synthetic oligopeptides are shown in Table2.

                  TABLE 2                                                         ______________________________________                                        REACTIVITY OF αF1 and αF2 WITH SYNTHESIZED                          α AND β PEPTIDES                                                   BOUND (OD.sub.490) MONOCLONAL ANTIBODY                                          Peptide*   αF1                                                                            αF2                                                                              βF1                                                                           δTCS1                               ______________________________________                                        α21-42                                                                             0.04   0.08       0.04 0.04                                          α141-159 0.26 0.05 0.08 0.04                                            α163-182 0.04 0.05 0.05 0.05                                            α212-231 0.03 0.86 0.06 0.05                                            β42-56 0.04 0.07 0.04 0.04                                               β144-161 0.05 0.05 0.04 0.06                                             β192-210 0.08 0.04 0.07 0.03                                             β231-244 0.06 0.04 0.06 0.04                                           ______________________________________                                         *α and β peptides from both the variable and constant region       were coupled to protein carrier and coated on the microtitre plates.          Reactivity with mAb was observed by developing a signal with a second goa     antimouse-HRP conjugate. The number after α and β indicate         their spanning location residue within their corresponding α and        β TCR chains.                                                       

mAb αF1 reacted with the oligopeptide derived from TCR α chain spanningfrom amino acid 141 to 159 (α141-159) whereas mAb αF2 reacted with adifferent oligopeptide derived from TCRα amino acid 212 to 231(α212-231). Both oligopeptides reside within the constant region of thea chain sequence. Six other oligopeptides derived from other regions ofα and β chains of human TCR did not react with either αF1 or αF2.Neither of the control antibodies, βF1 and δTCS-1, reacted with thesynthetic α or β chain oligopeptides.

In addition, in a competitive TCR-ELISA immunoassay, oligopeptideα141-159 blocked the binding of mAb αF1 to TCR isolated from HPB, with aK_(d) of 2×10⁻⁹ M (FIG. 4a). Similarly, synthetic oligopeptide α212-231was able to compete with the TCR of HPB for the binding to αF2 (K_(d)5×10⁻⁹ M) (FIG. 4b). None of the other oligopeptides tested was able tocompete with HPB TCR in the TCR-ELISA assay.

6.2.4. IMMUNOPRECIPITATION OF IN VITRO SYNTHESIZED α CHAIN TCR BY αF1AND αF2

To further confirm the specific reaction of αF1 and αF2 with a chain TCRprotein, both mAb were used in an immunoprecipitation assay using an invitro translated protein mixture containing a known α chain TCR proteinand an irrelevant protein, rabbit globin (FIG. 5, Lane 6). In thisassay, αF1 and αF2 specifically immunoprecipitated an α chain TCRprotein of 32 kD but not the irrelevant rabbit globin (12 kD) (FIG. 5,Lane 1 and 2). In contrast, there were no proteins immunoprecipitatedwhen two isotype matched, irrelevant mAb were used (FIG. 5, Lane 3 and4).

6.3. DISCUSSION

We have generated two mAb, αF1 and αF2, directed against two distinctepitopes on the constant region of the α chain of TCR protein. Both mAbwere shown to immunoprecipitate an αβ heterodimer of human TCR from αβ Tcell lines, such as HPB and Jurkat, as well as peripheral bloodlymphocytes. By immunohistological staining, both antibodies recognizedgreater than 90% of both the mature peripheral resting and activated Tcells and lymphocytes in thymus and tonsil. Both mAb alsoimmunoprecipitated the in vitro translated product of an α cDNA clone.Using synthetic oligopeptides prepared according to the publishedsequences of α chain constant region, we have identified the specificepitopes recognized by both mAb αF1 and αF2.

Both αF1 and αF2 do not stain viable T cells. It is surprising that evenαF1, which recognized an epitope in the constant region that was veryclose to the variable region is not able to recognize the surface TCRproteins on viable T cells. Thus, it appears that the epitopesrecognized by αF1 and αF2 on the constant region of α chain TCR arehidden on the cell surface. It should also be noted that αF1 stains allthe available T cells expressing the α chain TCR protein. Thisobservation indicates that the epitope recognized by αF1 is conservedamong all the αβ T cells.

TCR is present on the cell surface as a complex associated with at leastthree other proteins, the CD3 complex (Davis and Bjorkman, 1988, supra).Both the constant region of the αβ TCR and the CD3 complex are highlynon-polymorphic. This conservation of sequence suggests that theconstant region of TCR may serve as an important functional domain tointeract with the CD3 complex for the transduction of signals during Tcell activation. Upon binding of antigen, the variable region of TCR mayundergo conformational changes and subsequently alter the interactionbetween the constant region of TCR and the CD3 complex. The latteralteration may thus initiate the functional role of the CD3 complex insignal transduction during T cell activation. αF1 and αF2 will be usefulin further analyzing this process.

Both αF1 and αF2 are useful for studying TCR biosynthesis during T celldifferentiation. αF1 identifies T cells in tissue section, as does βF1.Numerous studies have suggested that in the thymus, during T celldifferentiation, TCR δ, γ and β chain genes are rearranged andtranscribed earlier than α chain genes (Chien et al., 1984, Nature312:31; Raulet et al., Nature 314:103; Snodgrass et al., 1985, Nature315:232). It is also suggested that in thymus T cells differentiate andmature gradually along their path from cortex to medulla and finally toperipheral blood. The differential staining patterns of αF1 versus βF1in the cortical area of the thymus may indicate the presence of such aprocess of T cell maturation.

αF1 and αF2 can also be used to study TCRα protein expression. It hasbeen reported that in the absence of α chain TCR, the CEM cell line doesnot express TCR and CD3 complexes on its cell surface (Uppenkamp et al.,1988, J. Immunol. 140:2801). However, upon stimulation with PMA, the CEMline does express surface CD3 complexes (Shackelford et al., 1987, J.Immunol. 138:613). Using αF1, we found that the unstimulated CEM line isnegative for the expression of α chain TCR protein. However, in thepresence of PMA, the intracellular level of α chain TCR proteinincreases and the TCR:CD3 complex appears on the cell surface. Thisobservation demonstrates that PMA induces the expression of a chainprotein, resulting in surface CD3 expression.

αF1 and αF2 can also be useful in the clinical classification ordiagnosis of lymphoid malignant or non-malignant disease. In mouse, ithas been demonstrated that the expression of β protein precedes that ofα protein. It is, therefore, possible to identify undifferentiatedleukemic cells where the various stages of differentation can besubclassified. Thus, αF1 and αF2 are useful reagents in analyzing thepathway of TCR expression in man and may also be valuable reagents forthe clinical classification of T lymphocytes in disease states.

Similarly, abnormal expression of TCRα in malignant cells can bedetected by αF1 and αF2. The T-cell lymphoma cell line SUP-T1 (Denny etal., 1986, Nature 320:549) expresses a hybrid mRNA transcript comprisedof an immunoglobulin heavy-chain variable region and a portion of TCRα,including J segment sequences. αF1 and αF2 were used to show that thishybrid mRNA is, in fact, translated into a protein which includes aportion of the TCRα constant region.

7. EXAMPLE: MONOCLONAL ANTIBODIES TO THE VARIABLE REGIONS OF HUMAN TCELL ANTIGEN RECEPTOR

7.1. RESULTS

7.1.1. GENERATION OF W112 AND 2D1 MONOCLONAL ANTIBODIES

To make mAb specific to Vβ5.3 and Vβ8.1, two human T cell leukemiclines, HPB and Jurkat, were used as immunogens. HPB expresses Vβ5.3 onits cell surface (Leiden and Strominger, 1986, Proc. Natl. Acad. Sci.USA 83:4456-4460; Leiden et al., 1986, Mol. Cell Biol. 6:3207-3214;Toyonaga and Mak, 1987, Ann. Rev. Immunol. 5:585-620), and Jurkat cellsexpress Vβ8.1 (Yanagi et al., 1984, Nature 308:145-149; Leiden andStrominger,1986, supra; Toyonaga and Mak, 1987, supra). BALB/c mice wereimmunized with 1×10⁷ HPB-MLT or Jurkat cells. Four weeks later, the sameamount of cells were injected intravenously into the primed mice and thesplenocytes were fused after four days with mouse myeloma P3X63Ag8.653cells. Ten days later, hybridomas were screened for anti-TCR activitywith an ELISA protocol as illustrated in FIG. 6(a), using HPB and Jurkatcell lysates. Positive wells were cloned by limiting dilution and clonesrescreened. Data from a sample ELISA rescreening is shown in Table 3.Out of 500 hybridoma supernatants screened, only 2D1 was positive in theELISA assay. Anti-TCR major-framework mAb, W76, was used as a positivecontrol as it binds to the constant region of every β chain. Anti-CD3mAb, OKT3, was used as the negative control.

                  TABLE 3                                                         ______________________________________                                        Screening:                                                                              2D1          W76    OKT3                                            ______________________________________                                        1         0.132        0.165  0.055                                             2 0.278 0.315 0.115                                                         ______________________________________                                         W76 is an antiTCR major framework reagent used as a positive control.         OKT3 is an antiCD3 reagent used as a negative control.                   

To further confirm the TCR-ELISA screening results, CD3-comodulationassays were run as shown in FIG. 6(b). For the comodulation assays,Jurkat or HPB cells were incubated overnight with hybridomasupernatants. Cells were then washed and reincubated withFITC-conjugated OKT3, and analyzed by flow cytometry. Positive hybridomasupernatants such as 2D1 that were able to modulate the TCR-CD3 complexoff of the cell surface, stained negatively with OKT3 the following day.Negative hybridoma supernatants stained positively with OKT3 thefollowing day. From the screening, mAb W112 reactive with HPB and 2D1reactive with Jurkat were identified.

7.1.2. SPECIFICITY OF W112 AND 2D1

The reactivity of W112 and 2D1 with TCR on HPB and Jurkat cells wastested using immunoprecipitation. ¹²⁵ I surface labelled HPB and Jurkatcell lysates were incubated with mAb and the precipitated immunecomplexes were then run on 10% SDS-PAGE (FIG. 7). W112 specificallyimmunoprecipitated a heterodimer of 48 kD and 40 kD from HPB lysates(FIG. 7A). 2D1 immunoprecipitated the same sized heterodimer from Jurkatcell lysates (FIG. 7B). The molecular weights of the immunoprecipitatedproteins are similar to those precipitated by βF1, a mAb reactive with aframework epitope of the β chain constant region, or by αF1, theanti-major framework anti-TCRα antibody (Section 6, supra).

The reactivity of W112 was also tested by Western blot analysis (FIG.8). HPB cell lysates were fractionated by 10% SDS-PAGE and thentransferred to nitrocellulose sheets. The sheets were first incubatedwith W112, washed and then incubated with horse radish peroxidase (HRP)conjugated goat anti-mouse antibody. After washing, color was developedwith an HRP substrate. W112 specifically reacted with a 40 kD protein.This 40 kD protein also reacted with the β constant region reagent βF1(FIG. 8) indicating W112 was TCRβ chain specific.

To determine the chain specificity of 2D1, Jurkat cells weremetabolically labelled with ³⁵ S-methionine and the cell lysates wereincubated with 2D1. Immunoprecipitated complexes were then fractionatedby 10% SDS-PAGE and the dried gel autoradiographed. Antibodies 2D1 andβF1 reacted with the 40 kD β chain of Jurkat cells (FIG. 9).

7.1.3. FLUORESCENT ACTIVATED CELL SORTING USING W112 AND 2D1

Reactivity of W112 and 2D1 was determined by FACS analysis. PBLs fromnormal donors were reacted with W112, 2D1, OKT3 or normal mouse serum,washed, incubated with FITC-labelled goat anti-mouse antibody andanalyzed on an Ortho® flow cytometer (FIG. 10). When tested againstnormal PBLs, W112 and 2D1 antibodies detected only minor populations ofcells. Among 10 samples tested, W112 reacted with from 0.3% to 5%peripheral blood cells, and 2D1 reacted with 0.5% to 13%. The positivecontrol antibody, OKT3, represented a pan-T cell reagent, and stained70% of the peripheral blood lymphocytes. The negative control reagent,normal mouse serum, was non-reactive. Since W112 and 2D1 react withsubsets of T cells, they are anti-minor framework antibodies.

7.2. DISCUSSION

Together these results indicate that both W112 and 2D1 antibodies detecta small subset of T cells in the large population of cells present inperipheral blood. W112 and 2D1 are TCR anti-minor framework specificreagents that react with only that subpopulation of cells in peripheralblood that expresses that particular minor framework determinantprotein. Both antibodies are specific to the β chain of the TCRheterodimer and furthermore are specific to that variable region presenton the cells used as their immunogen.

2D1 is a TCR Vβ8.1 specific reagent. W112 was determined to be a TCRVβ5.3 specific reagent by the following criteria. It has been reportedthat the T cell leukemia line HPB-MLT rearranges two TCR β genes whichare expressed at the mRNA level (Leiden, et al., 1986, Molec. Cell.Biol. 6:3207). Cloning and sequencing of cDNA clones corresponding tothe two rearranged genes revealed that one of the rearrangements isdefective and is characterized by a out-of-frame V-D-J joining eventwhich causes several stop codons in downstream C-region sequences. Asecond cDNA represents the functional β gene from HPB since it iscomposed of a full-length open reading frame and its deduced amino acidsequence corresponds to the amino-terminal protein sequence of the βchain isolated from HPB-ALL cells (Jones, et al., 1985, Science 227:311.The deduced amino acid sequence for the defective gene is identical tothe V region sequence which identifies the Vβ6.1 family (Concannon, etal., 1986, Proc. Natl. Acad. Sci. 83:6598; Kimura et al., 1986, J. Exp.Med. 164:739). The amino acid sequence for the functional gene isidentical to the V region sequence which identifies the Vβ5.3 family.Thus, TCR Vβ-specific antibodies which bind to HPB cells must recognizethe Vβ5.3 family. Thus, W112 is a TCR Vβ5.3 specific reagent.

W112 and 2D1 were generated against specific T cell clones havingdefined TCR proteins and selected for the preferred IgG isotype usingthe TCR ELISA. Different IgG subtypes for these antibodies could beselected by the isotype switching protocol described in Section 5.2,supra. These reagents have clinically relevant uses in disease diagnosisand therapy. Since these antibodies react with anti-minor frameworkdeterminants present on specific T cell subsets, they would be preferredclinical reagents for detecting or modulating Vβ5.3 and Vβ8.1 diseasespecific T cells as compared to anti-major framework reagents or pan-Treagents that by their nature must affect large populations of T cells.

The data presented supra for generating TCR antibodies of definedspecificity using whole T cell clones as immunogens and selecting forTCR reagents of defined isotype, can readily be extended to producingadditional anti-minor framework monoclonal antibodies. Antibodies toVβ12.1, Vβ1.2, Vβ12.4, Vβ2, Vβ9, and Vβ10 are being generated byimmunizing with the T cell clones or cell lines listed in Table 4. αβ⁺ Tcell clones can likewise be used to produce anti-minor framework orviable region specific anti-γ and anti-δ reagents.

                  TABLE 4                                                         ______________________________________                                        Cell line   Vβ                                                                              Vα    Dβ                                                                           Jβ                                    ______________________________________                                        C11         2.?                                                                 M11 2.2  2.1 2.3                                                              DT259 3.2  1.1 2.1                                                            2G2 4.1  2.1 2.5                                                              DT110 4.2  1.1 1.1                                                            HPB 6.1 12.1 2.1 2.5                                                          3A1, 5A5 6.1                                                                  ATL122 6.3 1.1 1.5                                                            L17 6.9 17.1 1.1 1.5                                                          Jurkat 8.1 1.2 1.1 1.2                                                        CEM2 9.1   2.3                                                                ATL121 10.2  1.1 1.5                                                          CEM1 12.4                                                                     ATL21 15.1  1.1 1.5                                                           HUT102 20.1   1.2                                                             DT55  4.3                                                                     SUPT1  7.2                                                                    HUT 78 18.1 1.                                                                MOLT 16 1. 2.                                                               ______________________________________                                    

8. EXAMPLE: ELEVATION OF A γδ T-CELL SUBSET IN PERIPHERAL BLOOD ANDSYNOVIAL FLUID OF PATIENTS WITH RHEUMATOID ARTHRITIS

The levels of δTCS1 positive and TCRδ1 positive T cells were determinedin the peripheral blood and synovial fluid of patients with rheumatoidarthritis (RA) and Felty's syndrome (FS). Elevated levels of a γδ T cellsubset identified by δTCS1 was found in the peripheral blood andsynovial fluid of patients with RA as well as in the peripheral blood ofpatients with FS.

8.1. MATERIALS AND METHODS

8.1.1. PATIENT SELECTION

Twenty-nine patients with definite or classical RA and 11 additionalpatients with Felty's Syndrome seen in the Rheumatic Disease Unit of theWellesley Hospital were studied. Controls consisted of 22 healthyvolunteers (NML) from the Wellesley Hospital and T Cell Sciences, Inc.Twelve of the controls were age-matched. Since the data obtained fromthe age-matched control subjects from the two groups were comparable tothose not matched for age, all control data was pooled. In addition, 5patients with seronegative spondyloarthropathies (3 with psoriasis, 1with Reiter's syndrome and 1 with ankylosing spondylitis) were examined.All patients were receiving nonsteroidal anti-inflammatory drugs(NSAIDs). In addition to NSAIDs, some of the RA patients had received atvarious times during active disease stages any of the following:remittive agents, prednisone, or cytotoxic agents/remittive agents.

8.1.2. CLINICAL EVALUATION

A number of clinical variables were examined in each patient,including: 1) disease duration, 2) number of actively inflamed joints(defined as those with tenderness or effusions), 3) erythrocytesedimentation rate (ESR; Westergren) and 4) medications.

8.1.3. PREPARATION OF MONONUCLEAR CELLS FROM PERIPHERAL BLOOD

Equal volumes of whole blood (containing anti-coagulant) andSepracell-MN (Sepratech Corp.) were added to a centrifuge tube. Aftergentle mixing, the tube was centrifuged at 1500×g at room temperaturefor 20 minutes. After density separation, mononuclear cells found in theopalescent compact band just below the meniscus were collected. Theywere washed twice by mixing the cells with four volumes of PBS-BSA (0.1%w/v), and subjecting the mixture to centrifugation at 300×g for 10minutes per wash.

8.1.4. IMMUNOFLUORESCENCE STAINING OF CELL SURFACE MARKERS

All patient cells were phenotyped by flow cytometry using the OrthoDiagnostics Cytofluorograph II (Ortho Diagnostic Systems, Inc., Raritan,N.J.). Fluorescein-conjugated monoclonal antibodies specific for variouscell surface determinants were used for a direct immunofluorescencestaining procedure. Briefly, 2-5×10⁵ mononuclear cells were suspended in100 μl of PBS with 0.2% bovine serum albumin and 0.05% sodium azide(flow buffer) at 4° C. Conjugated monoclonal antibodies (100 ng) wereadded to the cell suspension mixed well and incubated for 30 minutes at4° C. The stained cells were washed with flow buffer two times andfinally resuspended in the same buffer for cytometric analysis. Themonoclonal antibodies utilized for immunofluorescence included thefollowing from Ortho Diagnostics Systems, Inc. (Raritan, N.J.), OKT3,OKT4, and OKT8, which recognize the CD3, CD4 and CD8 determinants,respectively, and TCRδ1 and δTCS1, which recognize all human γδ T cellsand a subset of them, respectively.

8.2. RESULTS

We first examined the levels of TCRδ1⁺ T cells and δTCS1⁺ cells in theperipheral blood of patients with rheumatoid arthritis (RA) and comparedthem to the levels in peripheral blood of patients with Felty'ssynodrome (FS) and healthy control subjects (NML). The results (FIG. 11,left panel) revealed comparable log mean levels of peripheral bloodTCRδ1⁺ T cells in RA (5.2%) and NML (3.9%) patients (p>0.05), whereas FSpatients exhibited higher levels (6.8%) relative to the other groups;however, the difference was not statistically significant (p>0.05). Incontrast, mean levels of PB δTCS1⁺ T cells in RA (1.8%) and FS (4.5%)were significantly elevated relative to NML (0.8%) (p=0.007 and p=0.006,respectively) (FIG. 11, right panel). The level of δTCS1⁺ T cells in RAwas not statistically different from that in FS (p>0.05).

The level of peripheral blood δTCS1⁺ T cells was next examined as aproportion of TCRδ1⁺ cells. The results (FIG. 12) revealed an elevationin the mean ratio of δTCS1/TCRδ1 in both RA (0.4) and FS (0.6) relativeto NML (0.2) (p=0.02 and p=0.005, respectively). Moreover, in patientswith FS who demonstrated elevated levels of TCRδ1⁺ T cells, δTCS1⁺ Tcells accounted for the majority of TCRδ1⁺ T cells while in normals,δTCS1 T cells account for <20% of the TCRδ1⁺ T cell population. Theseresults suggested a correlation between the level of δTCS1⁺ T cells andTCRδ1⁺ T cells. Indeed, the level of δTCS1⁺ T cells correlated stronglywith the level of TCRδ1⁺ T cells in FS (r=0.98, p=0.001) (FIG. 13).

Since elevated levels of peripheral blood δTCS1⁺ T cells were observedin RA, we next asked whether the increased peripheral blood levels couldbe a reflection of a local increase in δTCS1⁺ T cells within thesynovium. To address this issue, we evaluated the levels of δTCS1⁺ Tcells and TCRδ1⁺ T cells in paired synovial fluid and peripheral bloodsamples from 16 RA patients and compared these levels to synovial fluidsamples from 9 patients with seronegative spondyloarthropathies (SSA),five of whom also had peripheral blood levels determined. The results(FIG. 14, lower panel) revealed comparable log mean levels of δTCS1⁺cells in paired RA peripheral blood and synovial fluid samples (1.9% and2.2%, respectively). Moreover, the level of δTCS1⁺ T cells in RAperipheral blood was elevated relative to the peripheral blood level inSSA patients (0.05%) (p=0.05), and there appeared to be a differencebetween the synovial fluid levels in RA and SSA (p=0.1). The level in RAperipheral blood and RA synovial fluid, but not in SSA synovial fluid,was greater than that in the peripheral blood NML group. The levels ofTCRδ1⁺ T cells in paired RA peripheral blood and synovial fluid sampleswere comparable (5.2% vs 5.2%) and not significantly different from thatin peripheral blood of NML controls (3.6%) (p>0.05 for both comparisons(FIG. 14, upper panel). Again, there was a trend toward a lower synovialfluid level in SSA (3.0%) relative to RA peripheral blood and synovialfluid levels.

When we examined δTCS1/TCRδ1 ratios in paired peripheral blood andsynovial fluid samples, we observed comparable ratios in RA peripheralblood (0.40), RA synovial fluid (0.5) and SSA synovial fluid (0.6),(p>0.05 for all comparisons) (FIG. 15). However, the δTCS1/TCRδ1 ratiosin RA synovial fluid were significantly elevated relative to that in NMLperipheral blood (0.2±0.5) (p=0.005). Of note, the δTCS1/TCRδ1 ratio inSSA synovial fluid was also elevated relative to NML peripheral blood(p=0.04). The ratio in SSA peripheral blood was comparable to those inNML peripheral blood and RA peripheral blood (p>0.05 for both).

8.3. DISCUSSION

The results of the present study demonstrate elevated levels of a γδ Tcell subset in the peripheral blood and synovial fluid of RA patientsand in peripheral blood of patients with FS. In contrast to thepreliminary data of Brennan, F. M., et al. (1988, J. Autoimmunity, 1,319-326) we did not observe an elevation in total γδ T cells in thesynovial fluid of RA patients. Brennan et al., however, examined pairedperiphral blood and synovial fluid samples from only 3 patients.

The pathogenic significance of an elevation in a γδ T cell subset in RAremains unclear. However, an increase in a T cell population in RAsynovial fluid with cytotoxic potential is consistent with theobservation of spontaneous cytotoxicity of synovial T cells directedagainst both self and non-self epitopes in RA (Example 9, infra). Thesefindings may not reflect non-specific recruitment of T cells into theinflammatory process; an elevation of a specific subset of γδ T cellswas observed, not an elevation in the whole γδ population. Moreover, theelevation of δTCS1⁺ T cells in RA synovial fluid and not SSA synovialfluid suggests that the increase in δTCS1⁺ T cells in RA is not a resultof non-specific effects of inflammation.

To date, a total patient population of 20 normals, 16 RAs, and 10Felty's patients has been evaluated for the presence of an elevatedδTCS1⁺ subset in peripheral blood. An elevated level, defined as themean value observed for the patients plus 2 standard deviations, wasobserved in 1/20 normals (5%), 4/16 RAs (25%) and 5/10 Felty's (50%).The proportion of patients with elevated δTCS1⁺ cells in the FS groupand RA population was statistically greater than that in the group ofhealthy subjects with X² =8.9 (p=0.003) and 2.9 (p=0.08), respectively.It is expected that as the general patient population is further dividedinto subgroups based upon HLA expression and disease state, that theselevels of correlation between δTCS1⁺ cells and disease will increase.

9. EXAMPLE: NOVEL AUTOREACTIVE CYTOTOXIC ACTIVITY AGAINST SYNOVIOCYTESBY RHEUMATOID ARTHRITIS DERIVED T CELL LINES AND CLONES

T cell lines were established from synovial tissue and peripheral bloodof RA and non-RA patients. Phenotypic and functional studies indicatedthat specific T cell recruitment occurs in reheumatoid arthritis joints.

9.1. MATERIALS AND METHODS

9.1.1. PATIENT SAMPLES

A total of 23 paired RA patient synovial tissue and peripheral bloodsamples were collected for this study. All the patients had seropositiveclassic RA with ages ranging from 38 to 77 years with a mean age of59.5. In addition to the RA patient samples, we also obtained tissuesamples from 8 non-RA patients. Seven of these patients had traumaticarthritis of the knee (i.e., torn meniscus) and their synovial tissuewas derived by arthroscopy. One of the eight (NST-7) non-RA tissuespecimens was from the knee of a patient with ankylosing spondilitis. Inthe non-RA patient group, a peripheral blood sample was obtained onlyfrom the ankylosing spondylitis patient.

9.1.2. SAMPLE PROCESSING AND CELL LINES

A panel of various cell lines were derived from each patient, wheneverpossible. T cell lines were obtained from both the disease site and theperipheral blood.

9.1.3. SYNOVIAL TISSUE-DERIVED T CELL LINES

Synovial tissue-derived T cell lines from RA patients (ST-line) wereinitially established by placing finely minced synovium in a 24 wellplate (Costar, Cambridge, Mass.) containing IL-2 medium which consistedof RPMI 1640 supplemented with 10% fetal calf serum (Hyclone, Logan,Utah.), 100 Units/ml penicillin, 100 ug/ml streptomycin, 1 mM sodiumpyruvate, 50 ng/ml gentamycin, 10 mM Hepes and 20 Units/ml ofrecombinant interleukin-2 (Il-2) (Amgen, Thousand Oaks, Calif.). Afterthe culture conditions had been optimized, the minced synovium wasinstead placed into fetal calf serum free medium consisting of AIM-V(Gibco, Grand Island, N.Y.), 5% human AB serum (Center for DiagnosticProducts, Milford, Mass.), 5% LymphocultT (Biotest, Fairfield, N.J.) and20 u/ml recombinant IL-2. The activated T cells within the diseasedsynovium migrated out of the tissue in the presence of IL-2 medium. TheST-line T cells were maintained by replacing half of the culture volumewith fresh IL2 medium twice a week. After 3 weeks in culture, the Tcells required additional stimulation. Initial cultures were stimulatedwith allogeneic, irradiated feeder cells from normal peripheral bloodlymphocytes (PBL) at a concentration of 2×10⁵ /well and 100 ng/ml of aCD3-specific monoclonal antibody, OKT3. ST-line T cells were cloned bylimiting dilution in round bottom 96 well plates (Costar, Cambridge,Mass.) with 1×10⁵ feeder cells per well. After optimization, latercultures were stimulated by growing the T cells on CD3 coated 24 wellplates (coated with 2.5 μg/ml OKT3) for a minimum of 3 hours, followedby washing 3 times and then plating the cells. It was observed that theuse of allogeneic and even autologous feeder cells biased the cellgrowth toward CD8+ cells during culture. The lines maintained theiroriginal phenotype better on CD3 coated plates.

9.1.4. PERIPHERAL BLOOD-DERIVED T CELL LINES

Peripheral blood-derived T cell lines (PB-T) were obtained by culturingPBL in IL-2 medium plus 1 ug/ml PHA (Welcome Research Laboratories,Beckenham, England).

9.1.5. B CELL LINES

B cell lines were derived from PBL by Epstein-Barr virus (EBV)transformation (Alpert, S. D., et al., 1987, J. Immunol., 138, 104).

9.1.6. PERIPHERAL BLOOD MACROPHAGES

Peripheral blood macrophages (PBMO) were obtained by overnight adheranceof PBL in RPMI medium plus 10% human AB serum (Center for DiagnosticProducts, Milford, Mass.). The non-adherant cells were thoroughly washedaway and the adherant macrophages isolated by incubation with ice coldcalcium/magnesium free phosphate buffered saline (PBS).

9.1.7. SYNOVIOCYTES

Synoviocytes were also obtained by culturing in IL-2 medium in either100 mm dishes in which the tissue was minced or in 6 well dishes(Costar, Cambridge, Mass.). The medium was usually supplemented withhuman serum unless otherwise stated. Type B synoviocytes were fibroblastcells which were negative for nonspecific esterase (Sigma, St. Louis,Mo.) and HLA-DR while Type A synoviocytes were nonspecific esterase andHLA-DR positive (Carson, D. A. and Fox, R. I., 1985, In "Arthritis andAllied Conditions", McCarty, D. J. (Ed.), p. 257; Iguchi, T., et al.,1986, Arth. Rheum., 29, 600). Separation of Type A and Type B cells wasaccomplished by differential adherance and trypsin sensitivity. Synovialfibroblast cultures could be obtained by 3-5 minute trypsinization froma tissue monolayer outgrowth or by allowing the fibroblasts to overgrowthe primary mixed culture. The mixed cultures that had 3-5 minutetrypsin treatment resulted in greater than 95% pure Type A,macrophage-like cultures, as measured by esterase staining. Not allpatient samples generated Type A cells in adequate numbers forfunctional assays. Both types of synoviocytes, PB macrophages and theEBV-B cell lines were used as autologous targets in cytotoxicity assays.

9.1.8. CELL SURFACE PHENOTYPING

All patient cell lines were phenotyped by flow cytometry using the OrthoDiagnostics Cytofluorograf II. Fluorescein-conjugated monoclonalantibodies specific for various cell surface determinants were used fora direct immunofluorescence staining procedure. Briefly, 2-5×10⁵ cellswere suspended in 100 μl of PBS with 0.2% bovine serum albumin and 0.1%sodium azide (flow buffer) at 4° C. Conjugated monoclonal antibodies(100 ng) were added to the cell suspension mixed well and incubated for30 minutes at 4° C. The stained cells were washed with flow buffer twotimes and finally resuspended in the same buffer for cytometricanalysis. The monoclonal antibodies utilized for T cell phenotypinginclude the following from Ortho, Raritan, N.J.; OKT3, OKT4, KT8, whichrecognize the CD3, CD4 and CD8 determinants, respectively. In additionto the T4/T8 subsets, we also stained for the helper-inducer andhelper-suppressor subsets using phycoerythrin-conjugated monoclonals 4B4and 2H4 (Coulter, Hialeah, Fla.). Finally, T cells were also stainedwith a V.sub.δ 1 TCAR specific monoclonal antibody, δTCS-1.Fluorescein-conjugated HLA DR Class II-specific antibody was purchasedfrom Becton Dickinson, Mountain View, Calif.

9.1.9. CYTOTOXICITY ASSAY

T cell mediated cytotoxicity was measured by a 3-hour ⁵¹ Cr releaseassay as previously described (Snider, M. E., et al., 1986,Transplantation, 42, 171). Briefly, target cells were labeled with50-100 μC ⁵¹ Cr for 30 minutes at 37° C. in a shaking water bath. Afterwashing the target cells three times in Hanks buffered saline containing10% serum, they were resuspended in assay medium which consisted of RPMIplus 10% fetal calf serum and 10 mM Hepes. Assay plates were prepared bythe addition of 200 μl volume of effector T cells adjusted to 6.25×10⁴per well (resulting in an Effector:Target ratio of 25:1) or by theaddition of 200 μl of medium or water alone. Radioactive target cellswere placed in prepared V-bottom 96 well plates (Costar, Cambridge,Mass.) at 2.5×10³ cells in a 20 μl volume. The plates were spun at 50×gfor 5 minutes and incubated at 37° C. in a humidified, 5% CO₂ incubatorfor 3 hours. The assay was harvested by removing 100 μl of supernatantfrom each quadruplicate well and radioactivity release was measured onan LKB gamma counter. Percent specific lysis was calculated using thefollowing formula: ##EQU1## where ER=mean ⁵¹ Cr release in the presenceof effector T cells, SR=mean spontaneous ⁵¹ Cr release in media alone,X=mean maximum ⁵¹ Cr release in water and B=machine background. Meanswere calculated from quadruplicate wells and standard deviations neverexceeded 10%.

9.1.10. IN SITU IMMUNOHISTOCHEMISTRY

Synovial tissue in about 2 cm square pieces was snap frozen in liquidnitrogen, coated with OCT media, and stored at -70° C. Frozen sectionswere cut at 5 μm on a cryostat, placed on microscope slides, air dried,and fixed in acetone for 5 minutes at room temperature. Sections wererehydrated in PBS and stained with δTCS1 at 10 μg/ml final concentrationfor 30 minutes at room temperature. Reactivity with δTCS1 antibody wasdetermined using a commercial immunperoxidase kit specific for mouse Ig(Ortho, Raritan, N.J.). Cells were counterstained using hematoxylin andexamined by light microscopy.

9.1.11. SUPPRESSOR FACTOR ASSAY

Normal peripheral blood lymphocytes were stimulated with either OKT3 orPHA. Suppressor activity was determined by the ability of a γδ⁺ cellculture supernatant to inhibit the proliferative response of PBLs toOKT3 or PHA. Culture supernatants were generated from RA synovial tissuederived T cell cultures or from the paired RA PBL derived T cellcultures following stimulation on either OKT3 or δTCS1 coated plates.Plates were coated first with 2.5 μg/ml goat anti-mouse IgG1 (SouthernBiotech Assoc., Birmingham, Ala.) followed by the appropriate monoclonalantibody at 2.5 ng/ml for 3 hours at 37° C. Control cultures receivedeither no stimulation or stimulation with irradiated allogeneicperipheral blood lymphocytes plus 100 ng/ml OKT3. Monoclonal antibodycoated plates were selected to eliminate the production of lymphokinesby the allogeneic feeders. Normal PBLs were plated in 96 well flatbottom plates at 8×10⁴ cells/well in RPMI 1640 plus 10% FCS with either10 ng/ml OKT3 or 0.5 ng/ml PHA (Wellcome Diagnostics, Dartford,England). Culture supernatants were added at a starting dilution of 1:4with serial dilutions up to 1:1024. Assays were pulsed on day 3 with 1.0μCi/well of tritium for 8 hours before harvesting.

9.2. RESULTS

9.2.1. CELL SURFACE PHENOTYPE OF SYNOVIAL TISSUE-DERIVED AND PERIPHERALBLOOD-DERIVED T CELLS

9.2.1.1. δTCS1 CELL SURFACE PHENOTYPE

The gamma delta T cell antigen receptor specific monoclonal antibody,δTCS1 (Wu, Y-J., et al., 1988, J. Immunol., 141, 1476-1479), was used toquantitate the gamma delta T cell receptor positive T cells infiltratingsynovial tissue. Table 5 compares the percentage of δTCS1 positive Tcells from RA patient synovium (ST-line) with the peripheral blood Tcells of each of the 23 paired RA samples and 8 paired non-RA samples.

                  TABLE 5                                                         ______________________________________                                        PERCENTAGE OF δTCS1 POSITIVE T CELLS                                      DERIVED FROM ARTHRITIS PATIENT T CELL LINES*                                    Patient Sample                                                                             Synovial Tissue                                                                           Peripheral Blood                                 ______________________________________                                        ST-2         12          5                                                      ST-9 7 1                                                                      ST-17 0 7                                                                     ST-18 2 1                                                                     ST-24 2 1                                                                     ST-25 64 11                                                                   ST-27 10 5                                                                    ST-28 18 8                                                                    ST-29 2 13                                                                    ST-30 0 0                                                                     ST-31 2 1                                                                     ST-32 34 0                                                                    ST-33 13 0                                                                    ST-34 1 1                                                                     ST-35 80 3                                                                    ST-36 1 2                                                                     ST-37 7 1                                                                     ST-38 5 0                                                                     ST-39 9 3                                                                     ST-43 8 0                                                                     ST-48 5 3                                                                     ST-49 1 5                                                                     ST-51 3 1                                                                   x ± SD    12.4 ± 20                                                                              3 ± 4                                             NST-1        2            NA.sup.+                                              NST-2 3 NA                                                                    NST-5 8 NA                                                                    NST-7 2 +                                                                     NST-9 2 NA                                                                    NST-10 9 NA                                                                   NST-17 5 NA                                                                   NST-13 1 NA                                                                 x ± SD    4 ± 3    NA                                                   ______________________________________                                         *= double immunofluorescence with OKT3 and δTCS1 monoclonal             antibodies. Numbers are expressed as percentage δTCS1 positive cell     relative to percentage CD3 positive cells, in order to standardize for        samples having varying amounts of CD3+ cells.                                 .sup.+ NA = not available (no peripheral blood received)                 

In normal subjects, gamma-delta T cells comprise 1-5% of peripheralblood T cells (Wu, Y-J., et al., supra; Brenner, M., et al., 1986,Nature, 322, 145). Our results show that while the values of percentageof δTCS1 positive T cells derived from peripheral blood derived T celllines from RA patients were within the normal range, the values forseveral paired RA synovial tissue lines were above the normal range(Table 5). Indeed, a marked increase in the proportion of gamma-delta Tcells was observed in RA synovial tissue lines. The lower half of Table5 demonstrates that in most cases the proportion of gamma delta cellsfrom non-RA synovial-derived T cells (NST-lines) are not significantlyelevated. These paired patients' T cell lines were cultured in parallel,with the mean length in culture of 18.8±8 days, ranging from 11 to 36days.

Three of the synovial tissue lines were stained by doubleimmunofluorescence with fluorescein-cinjugated OKT8 andphycoerythrin-conjugated δTCS1. We found a significant percentage ofgamma delta+ cells were also CD8+, since the percent of double stainingcells for ST-25, ST-32 and ST-33 were 47%, 23%, and 5%, respectively. Inaddition, the majority of the gamma delta+ cells in ST-35 were CD4+since the T cell line was 94% CD4+ and 80% δTCS1⁺. These findingssuggest that at least in some synovial tissue, the gamma delta+ cellsare not members of the CD4⁻ CD8⁻ cell population.

9.2.1.2. CD4, CD8, 4B4, 2H4 CELL SURFACE PHENOTYPES:

Synovial tissue lines and paired peripheral blood T cell lines fromseven RA patients different from those listed in Table 5 were analyzedfor T cell subset information (i.e. CD4/CD8 ratio andsuppressor-inducer/helper-inducer cell ratio). These paired RA patient Tcell lines were cultured in parallel with the mean length in culture of13±1.8 days, ranging from 9 to 15 days. The RA patients' lines were alsocompared with synovial tissue-derived T cells from four non-RA patients(NST-line). The NST-lines were in culture for a mean of 14±3 days,ranging from 10-19 days. Table 6 confirms previous reports (Forre, O.,et al., 1982, Scand. J. Immunol., 16, 815) that T cells derived fromdiseased joints and stimulated with PHA are predominantly CD4+ (meanvalue of 79%±15) and 4B4+ (mean value of 91.4%±6) while the T cells fromthe peripheral blood of the same patients had an equal mixture of CD4and CD8 populations (mean values for CD4 and CD8 were 43.8%±17 and48.8%±12, respectively).

                  TABLE 6                                                         ______________________________________                                        Comparison of Cell Surface Phenotype of Rheumatoid                              and Non-Rheumatoid T Cell Lines.*                                                   Phenotype Marker                                                                                                4B4/                                  Sample CD4 CD8 4B4 2H4 2H4                                                  ______________________________________                                        ST-LINE 79 ± 15                                                                             13.9 ± 8.6                                                                           91.4 ± 6                                                                           3.1 ± 1.8                                                                         29.5                                  ST-PBT 43.8 ± 17 48.8 ± 12.4 69.9 ± 14.6 22.2 ± 17 3.1                                                     NST-LINE 89 ± 6.5 6.7 ±                                                2.4 92.4 ± 8.4 1.4 ± 0.7                                                66                                  ______________________________________                                         *= % positive ± standard deviation                                    

However, this is the first reported evidence that the phenotype ofcultured T cells from synovial tissue of RA and non-RA patients aresimilar since the NST-line CD4 mean value was 89%±6 and the mean 4B4value was 92.4%±8. Again, the PBT cells from the RA patients had a morenormal distribution of helper-inducer and suppressor-inducer cells, ascompared to the infiltrated T cells (i.e. the mean values for ST and PBTwere 69.9%±15 4B4 and 22.2%±17 2H4). However, the ratio of 4B4/2H4 inthe RA patients was distinct from normal subjects regardless of thesource of T cells studied (Morimoto, C., et al., 1985, J. Immunol., 134,1508).

9.2.2. IN SITU IMMUNOHISTOCHEMISTRY

Samples of synovial tissue were analyzed by in situ staining with δTCS1to determine whether γδ⁺ cells were in fact present in inflamed RAjoints and that their detection was not due to the selective expansionof a minor T cell subset during the cell culture procedure. As can beseen in Table 7, δTCS1⁺ cells were present in the majority ofinfiltrated synovial membrane tissue.

                  TABLE 7                                                         ______________________________________                                        In situ Staining by T cell Specific Monoclonal                                  Antibodies in Untreated Synovial Tissue                                         Sample  DR    Infiltrate                                                                             CD3  CD4   TCRδ1                                                                         δTCS1                       ______________________________________                                        ST-46   -     +          +    +     -     -                                     ST-47 - ++ ++  + +                                                            ST-48 - ++ ++  + +/-                                                          ST-49 - - -  - -                                                              ST-50 - - -  - -                                                              ST-51 - ++ ++ ++ + +                                                          ST-52 - ++ ++  + +                                                            NB-02 4 +++ +++  - -                                                          NB-04 4 +++ +++  + +                                                          NB-05 4 +++ +++  +/- +/-                                                      TW-01 - +++ +++  + +                                                        ______________________________________                                    

Thus, δTCS1 cells have been detected in the peripheral blood, synovialfluid and synovial membrane of patients with RA and in the peripheralblood of Felty's patients.

9.2.3. FUNCTIONAL ACTIVITY OF SYNOVIAL TISSUE DERIVED T CELLS

9.2.3.1. CYTOTOXICITY ACTIVITY

Cytotoxicity of T cell lines from RA synovial tissue against theirautologous Type A synoviocytes (macrophage-like cells), Type Bsynoviocytes (fibroblasts) and peripheral blood macrophages was measuredin a 3 hour ⁵¹ Cr release assay (Table 8].

                  TABLE 8                                                         ______________________________________                                        Cytotoxicity of RA Synovial Derived T Cell Lines *                                Effector Target Cell                                                      Cell     Type A      Type B    Peripheral Blood                                 ST-Line: Synoviocytes Synoviocytes Macrophages                              ______________________________________                                        ST-1     80          9         ND                                               ST-9 50 5 5                                                                   ST-11 41 8 ND                                                                 ST-13 50 2 6                                                                  ST-14 42 4 24                                                                 ST-15 11 7 ND                                                                 ST-16 1 5 ND                                                                  ST-17 61 0 ND                                                                 ST-22 27 2 ND                                                                 ST-25 26 6 13                                                                 ST-28 0 7 4                                                                 ______________________________________                                         * = % specific lysis in 3 .sup.51 hour Cr release assay.                 

ST-lines derived from nine out of eleven patients demonstratedcytotoxicity against autologous Type A synovial target cells while notaffecting the viability of the Type B synovial cells or PB macophages.In addition, autologous B cells were not lysed by the ST-lines. Thenatural killer (NK)-like activity of the T cell lines was variable anddid not correspond with the lytic activity against autologous synovialmacrophage-like cells. In fact, cloning of T cells from a patient (ST-1)whose line exhibited high NK-like activity resulted in Type A-specificclones with no detectable NK-like activity (Table 9).

                  TABLE 9                                                         ______________________________________                                        Cytotoxic Activity of Synovial Tissue                                           Derived T Cell Line and Clones *                                                Effector                                                                    Cell Target Cells                                                           ST-Line  Type A        Type B                                                   or Clone: Synoviocytes Synoviocytes K562                                    ______________________________________                                        Line     81            9         63                                             Clone 1 58 0 8                                                                Clone 2 60 0 1                                                                Clone 3 32 0 2                                                                Clone 4 24 0 0                                                              ______________________________________                                         * = % specific lysis in 3 hour .sup.51 Cr release assay.                 

Table 9 also shows that both CD4+ and CD8+ clones could specificallylyse Type A synoviocytes since clones 1, 3 and 4 were CD4+ while clone 2was CD8+.

The specificity of ST-lines for their syngeneic tissue-derived targetswas not shared by the peripheral blood T cell lines from the samepatients. In fact, the peripheral blood derived T cells followed twopatterns of cell lysis: either they were very lytic against the entiretarget cell panel including K562, or they had very weak activity againstK562 and occasionally other target cells as well. An example of thelatter is shown in Table 10.

                  TABLE 10                                                        ______________________________________                                        Comparison of the Cytotoxic Activity of a                                       Synovial-Derived T Cell Line and a Peripheral                                 Blood T Cell from a Single Patient.                                                          % Specific Lysis By:*                                        TARGET CELLS     ST-Line  ST-PBT                                              ______________________________________                                        Type A Synoviocytes                                                                            50       3                                                     Type B Synoviocytes 2 4                                                       PB-Macrophage 6 21                                                            EBV-B Cell 0 0                                                                K562 11 18                                                                    Daudi 2 1                                                                   ______________________________________                                         *The effectorto-target cell ratio was 25:1 and this experiment was done       with the RA patient, ST13.                                               

In this case, one patient's (ST-13) ST-line and PB-T cytotoxicity wasmeasured against a panel of autologous target cells, K562, and thelymphokine-activated killer (LAK)-sensitive target cell, Daudi. TheST-line lysed the Type A cells and had some detectable natural killercell-like activity while not lysing any other target cells in the panel.However, the PB-T cells from the same patient which were cultured inparallel did not lyse either of the autologous synoviocytes but did havesome NK-like activity and lysed some of the PB macrophages. To examinethe specificity of ST-13 line activity more closely, a cold targetinhibition assay was performed (FIG. 16). Lysis of ⁵¹ Cr labeled Type Acells by ST-line T cells was directly inhibited by the addition ofincreasing numbers of unlabeled Type A cells. However, addition ofincreasing numbers of unlabeled K562 did not inhibit ⁵¹ Cr release ofthe Type A cells, suggesting that the lysis of Type A cells is not dueto NK-like activity.

The Type A cell specificity of RA synovial tissue-derived T cells wasnot shared by synovial-derived T cells from the one out of nine non-RApatients(NST-3) where cultures yielded enough Type A synoviocytes for ⁵¹Cr labeling. Table 11 shows the cytotoxic activity of 2 NST-lines and 2ST-lines against a panel of target cells including autologoussynoviocyte targets, allogeneic synoviocyte targets and allogeneic Bcells.

                  TABLE 11                                                        ______________________________________                                        Comparison of the Alloreactivity and Target Specificity by                      Non-Rheumatoid and Rheumatoid Arthritis-Derived T Cell Lines                               % Specific Lysis by:                                           Target Cells   NST-3   NST-7    ST-17 ST-22                                   ______________________________________                                        Autologous Type A cells                                                                      17      NA       61    27                                        Autologous Type B cells 42 NA 0 2                                             Allogeneic Type A cells 0 3 7 34                                              Allogeneic Type B cells 7 2 39 4                                              Allogeneic EBV-B cells 2 0 33 40                                            ______________________________________                                         The allogeneic synoviocyte and B cell targets were derived form Rheumatoi     Arthritis patients.                                                           NA = target cells not available                                          

NST-3 line T cells lysed Type B cells to a greater extent than the TypeA cells and neither NST-lines had any significant alloreactivity. Incontrast, the synovial tissue derived lines both appeared to lyse theType A cells while not affecting the Type B cells. In addition, the Tcells from these two patients demonstrated alloreactivity againstEpstein Barr Virus B cells and cytolytic activity against allogeneicType A cells for ST-22 or allogeneic Type B cells for ST-17. Thisalloreactivity was only seen in these 2 patients from the panel of 11patients tested to date.

9.2.3.2. CYTOTOXIC ACTIVITY OF A γδ POSITIVE T CELL LINE; EFFECT OFδTCS1 MONOCLONAL ANTIBODY

T cell line ST-25, derived from the synovial tissue of a rheumatoidarthritis patient, was assayed for cytotoxic activity against itsautologous target cells. The effector cells were pretreated with δTCS1(2 μg/ml) and then washed. The anti-HLA-DR (Becton Dickinson, MountainView, Calif.) and the anti-KLH (Keyhole limpet hemocyanin)controlantibodies were added at the initiation of the assay (2 ng/ml) and werepresent during the 3 hour assay. At the time of the assay, the phenotypeof ST-25 was 41.8% double positive for δTCS1 and CD3 and 39.3% doublepositive for δTCS1 and CD8. The results of the ST-25 killing assay aregiven in Table 12.

                  TABLE 12                                                        ______________________________________                                        Cytotoxicity Data for T Cell Line ST-25 *                                                                      ST-25  ST-25                                     ST-25 plus plus                                                               anti-HLA-DR δTCS1 anti-KLH                                            Target Cells ST-25 alone antibody antibody antibody                         ______________________________________                                        ST-25 Type A                                                                            17        7          52     15                                        Synoviocytes                                                                  ST-25 Type B                                                                  Synoviocytes 9 ND ND ND                                                       ST-25 B Cell 0 ND ND ND                                                       Line                                                                          ST-25 PBMO 24 14 124 43                                                       PB Line 13 0 0 4 ND                                                           PB Line 0 1 21 0                                                              K562 29 ND ND ND                                                            ______________________________________                                         * = % specific lysis at an effector target ratio of 25:1                      ND = not determined                                                      

The ST-25 cell line effectively killed ST-25 autologous Type Asynoviocytes and peripheral blood macrophages and K562 cells.Preincubation of ST-25 cell line with an anti-HLA-DR antibody (BectonDickinson, Mountain View, Conn.) caused a slight inhibition of theassay, while a control antibody to keyhole limpet hemocyanin (KLH)caused little effect. Treatment with δTCS1, however, resulted in asignificant enhancement of the lysis.

In summary, the activity of some subsets of γδ positive cells may becytotoxic and it may be possible to enhance the killing activity of thissubset by treatment with δTCS1 monoclonal antibody. This antibody alsoshows mitogenic activity (Wu, Y-J., et al., 1988, J. Immunol., 141,1476-1479). Using appropriate amounts of antibody, δTCS1 binds to thesurface of γδ positive cells and stimulates the proliferation of a minorcell population of resting human PBL to levels where the proportion ofγδ positive cells in the culture exceeds 90%.

9.2.3.3. SUPPRESSOR ACTIVITY OF γδ POSITIVE ST CELL LINES

For the suppression assays, fresh peripheral blood lymphocytes wereprepared and then stimulated with either PHA or anti-CD3 monoclonalantibody. The inhibition of the proliferation of these cells was testedby adding varying dilutions of supernatant fluid obtained from the cellcultures of ST-25 and ST-32 cell lines. Supernatant fluid from bothST-25 and ST-32 cell lines suppressed the proliferation of PHA oranti-CD3 stimulated PBLs. This suppression was observed at supernatantdilutions exceeding 1:1024 for ST-32 and 1:256 for ST-25. The factorsare soluble and are not removed by dialysis overnight at 4° C.(molecular weight cutoff of 3,000). The antibody, δTCS1, is mitogenicfor γδ⁺ cells (see 9.3.3.2, supra) and it may be that, following thetriggering of these cells with the antibody, suppressor factors areproduced. This would result in the ability to modulate this affect inthe γδ⁺ T cell subset by using the appropriate dosage of antibody.

9.2.4. BLOCKING OF THE CYTOTOXICITY REACTION BY δTCS1

To establish the ability of δTCS1 monoclonal antibody to block thecytotoxic activity of δTCS1⁺ effector cells against specific andnon-specific target cells, the RA derived effector cells may bepreincubated with δTCS1 antibody using a range of concentrations. Todistinguish between Fc receptor dependent killing and δTCS1 specificblocking in the cytotoxicity assays, both whole antibody and Fabfragments of δTCS1 antibody may be tested. The whole antibody may beused to give rise to Fc receptor mediated killing, whereas the Fabfragment (which has the Fc portion of the antibody removed) may be usedto effect specific blocking of γδ⁺ effector cell cytotoxic activity.

9.2.5. DEPLETION ASSAY USING δTCS1

To establish the ability of δTCS1 monoclonal antibody to modulate γδ⁺ Tcells and cause their specific elimination in vitro, δTCS1 may be testedin complement mediated lysis assays. For these assays, RA derivedeffector cells may be preincubated with δTCS1 antibody using a range ofconcentrations. Following preincubation, rabbit serum may be added as asource of complement pathway components. With the appropriateconcentrations of δTCS1, the complement cascade may become activatedleading to complement dependent lysis of the δTCS1⁺ cells and theirdepletion from the cell population. Effective in vivo concentrations ofδTCS1 for T cell elimination may be estimated to be approximately equalto those observed for the therapeutic monoclonal antibody, OKT3 (seeExample 10, infra).

9.3. DISCUSSION

The results of this study provide phenotypic and functional evidencethat specific T cell recruitment occurs in rheumatoid arthritis joints.It is well known that CD4+ T cells infiltrate the diseased synovium andare in close contact with HLA DR+ synoviocytes (Iguchi, T., et al.,1986, Arth. Rheum., 29, 600; Harris, E. D., Amer. J. Med., 80, 4). Thisclose association of T cells and HLA DR+ tissue macrophage-like cells isthought to be responsible for propagating the inflammatory response inthe synovium of RA patients. The exact nature, or even the existence ofspecific antigens causing RA is unclear. However, it is believed thatantigens may be presented by the synoviocytes with elevated HLA DR, theappropriate class II restricting element for CD4+ cells. Therefore, weisolated and expanded the diseased tissue-infiltrated T cells forphenotypic and functional characterization with a specific focus on theinteraction of those T cells and the other cell types found in thediseased, autologous synovium.

A novel functional response from synovial tissue-derived T cells wasobserved. Short-term cultured lines and cloned T cells from diseasedjoints were able to specifically lyse autologous Type A macrophage-likesynoviocytes, in vitro. Previous studies have shown that peripheralblood derived T cells could lyse synovial type B fibroblasts, however,this was observed primarily against allogeneic fibroblasts (Griffiths,M. M., et al., 1976, J. Clin. Invest., 58, 613; Person, D. A., et al.,1876, J. Clin. Invest., 58, 690). Our data represents the first reportof autologous Type A synoviocyte cytotoxicity by synovial tissue derivedT cells, and the lytic activity we observed in the PB-T lines was mostlyof an NK-like nature. The structure(s) recognized by the ST-lines andclones are not known; however, it does not appear to be class II antigenalone since DR+ autologous peripheral blood macrophages and B cells werenot killed in the same assays. Attempts are ongoing to isolate adequatenumbers of Type A cells for future molecular analysis of uniqueproteins. In the meantime, T cells exhibiting this unique pattern oftarget cell recognition are abundant.

Cell surface staining with a monoclonal antibody which recognized thesecond type of T cell antigen receptor, γδ cells, showed that RAsynovium contained significantly higher numbers of γδ T cells than thosefound in peripheral blood of the same patient. In addition, those gammadelta+ cells were not from a double negative CD4-CD8-population sincethey stained brightly for either CD8 or CD4 in those patients studied bydouble immunofluorescence. The selection of γδ+ T cells seen in RAsynovium but not in non-RA synovium has not been previously reported.The specificity and function of human γδ T cells is not yet fullyunderstood; however, preliminary reports suggest that they may beHLA-unrestricted killer cells (Faure, F., et al., 1988, J. Immunol.,140, 1372) and murine γδ cells are thought to be alloreactive (Maeda,K., et al., 1987, Proc. Natl. Acad. Sci. USA, 84, 6536; Matis, L. A., etal., 1987, Nature, 330, 262). Preliminary information on thecytotoxicity of the RA-derived γδ+ T cell lines indicates some directalloreactivity in ST-28 line while no detectable alloreactivity wasobserved with the others tested.

The synovial tissue of RA patients is known to be infiltrated with thehelper-inducer subset of T cells by immunofluorescence of frozenmembrane sections (Duke, O., et al., 1987, Arth. Rheum., 30, 849). Wehave confirmed this observation using isolated T cells obtained frompieces of synovium cultured in the presence of IL-2 medium. There issome discrepancy concerning the ratio of CD4+ to CD8+ cells in theperipheral blood of RA patients. Some reports indicate an increasednumber of CD4+ cells (Fox, R. I., et al., 1982, J. Immunol., 128, 351),others show a decrease in the CD4+ cells (Forre, O., et al., 1982,Scand. J. Immunol., 15, 221) and still others observe no change in the Tcell ratios in peripheral blood (Silverman, H. A., et al., 1976 Arth.Rheum., 19, 509). Our data demonstrates a decreased relative number ofCD4+ cells and an increased number of CD8+ cells as compared to thevalues found in PHA stimulated. peripheral blood, resulting in a lowerthan normal ratio of CD4/CD8 cells. In addition, we found a lower thannormal level of 2H4+ cells in the peripheral blood of RA patients;however, the defect in suppressor-inducer cells is more dramatic in thetissue than in the blood. Moreover, the major T cells involved in thepathogenesis of joint damage are the helper-inducer T cells and thenormal counterbalance provided by the suppressor-inducer cells waslacking in the joint and reduced in the periphery of RA patients.

Several reports have indicated that the T cell abnormalities in RAinclude diminished suppressor cell activity, decreased mitogenicresponses and increased antibody production (Silverman, H. Aa., et al.,1976, Arth. Rheuem., 19, 509; Indiveri, F., et al., 1986, Cell.Immunol., 97, 197; Wernick, R. Mm., et al., 1985, Aarth, Rheum., 28,742). Also, depressed lymphokine production and responsiveness have beenreported for both IL-2 and gamma-interferon (Lotz, M., et al., 1986, J.Immunol., 136, 3643; Husby, B. and Williams, R. C., 1985, Arth. Rheum.,28, 174). In most cases, however, T cell responses were not measured inthe context of synovial tissue-derived antigens. Our data demonstratedthe effector function against autologous synovial tissue-derivedantigens. The cytotoxicity does not appear to be mediated by NK-likemechanisms. Evidence for this tissue specificity in vivo is yet to bedetermined but the implication is that the infiltrating T cells may bedirectly contributing to the membrane damage in the disease process. Infact, the quantitative relationship between Type A and Type Bsynoviocytes in the synovial membrane is known to be altered in RA(Carson, D. A. and Fox, R. I., 1985, In "Arthritis and AlliedConditions", McCarty, D. J. (Ed.), p. 257). An increased number of TypeA cells early in the synovitis may even provide a mechanism of selectionin directed homing of T cells capable of recognizing Type A antigen(s).This hypothesis of T cell selection in the diseased joint is supportedby the finding that T cells in the PB of RA patients did not exhibit thesame pattern of target specificity as T cells cloned directly from thesynovium. Additional evidence for a disease-related T cell migrationinto the synovium may include the lack of Type A specificity in the Tcells derived from non-RA synovium, but more patient samples must firstbe tested.

In summary, we provide evidence of a novel functional specificitymediated by RA synovial-derived T cells. T cells from RA peripheralblood or non-RA synovium did not show any preferential target cell lysiswhich indicated that the unique structure(s) recognized by synovialtissue lines may be disease related. Many of the synovial tissue linesexhibited a significantly higher percentage of γδ+ T cell antigenreceptors as compared to either peripheral blood or non-RAsynovial-derived T cells. In contrast, the CD4/CD8 ratios and 4B4/2H4ratios were similar in ST-lines and NST-lines implying no diseasecorrelation with these classes of T cell subsets infiltrating thesynovial membrane. Because of this, the nature of the TCAR specificityshould be more informative than studying the surface CD4, CD8, 4B4, 2H4or similar surface phenotypes of T cell subsets. Most data to datesupport the hypothesis that RA patients, as well as other autoimmunepatients (Maeda, K., et al., 1987, Proc. Natl. Acad. Sci. USA, 84,6536), lack sufficient suppressor cells to control autoreactivity. Ourfindings are consistent with an aggressive, but novel, autoreactivityand depressed suppressor inducer T cells in RA patients. Studies areunderway to study the molecular nature of the T cell antigen receptorsfound in the diseased synovium (see Example 8, supra and Example 11,infra) and should provide information on the autoimmune mechanismspropagating chronic RA. The contribution of T cell-mediated Type Asynoviocyte destruction in RA or other inflammatory arthrophathiesremains unclear and is being studied further.

10. EXAMPLE: δTCS1, A MONOCLONAL ANTIBODY REACTIVE WITH THE Vδ1 REGIONOF HUMAN T CELL ANTIGEN RECEPTOR, IS USEFUL IN THE TREATMENT OFRHEUMATOID ARTHRITIS

Our results show that under different conditions, the antibody δTCS1 canspecifically modulate the activity of a subset of γδ⁺ T cells. Thesecells are elevated in the synovial fluid and peripheral blood of RApatients and in the peripheral blood of Felty's patients. In addition,they are present in the population of T cells that infiltrates theinflamed synovial tissue of RA patients. Improved therapies can bedeveloped using δTCS1, because this antibody can selectively modulate aspecific disease related subset of T cells and not affect other T cellpopulations. Depending upon the dosage of antibody used, δTCS1 basedimmunotherapies will either stimulate the specific T cell subset orcause it's elimination from the body; and enhance or block the T cellsubset's function. For anti-CD3 monoclonal antibody treatments,different elimination and stimulation therapies have been described(Chatenoud et al., 1982, Eur. J. Immunol. 12:979-982; Hirsch et al.,1989, J. Immunol. 142:737-743; Chatenoud et al., 1988, C. R. Acad. Sci.Paris, 307:833-836). The protocol given below as an example is onedesigned to specifically deplete δTCS1⁺ T cells from patients in whomtheir presence is deleterious.

10.1. PRECLINICAL DATA

10.1.1. PERIPHERAL BLOOD STUDY IN RA AND FELTY'S PATIENTS

During a preliminary study of the T cell subsets in RA patients in 1987,we observed a patient with extremely elevated δTCS1⁺ T cells (70%) inhis blood. Upon further inquiry, the patient was diagnosed to haveFelty's Syndrome. To date, our study of γδ+T cells in disease hasincluded 40 RA patients, 11 Felty's patients, 5 non-RA joint diseasepatients and 22 age matched normals. As shown in Example 8, 45.5% of theFelty's and 27.5% of the RA patients showed significant elevations ofδTCS1⁺ T cells (mean of normal plus 2 standard deviations) in peripheralblood.

The prevalence of various autoimmune diseases is associated with welldefined HLA phenotypes (see Table 13).

                  TABLE 13                                                        ______________________________________                                        Some HLA-DR-Associated Autoimmune Diseases                                                  HLA    PATIENT   NORMAL Relative                                  Disease Type % % Risk                                                       ______________________________________                                        Felty's   DR4    95          20     76                                          Syndrome                                                                      RA with DR4 95 20 76                                                          Vasculitis                                                                    RA DR4 68 25 3.8                                                              IDDM DR4 72 24 9.1                                                             DR3 49 22 4.3                                                              ______________________________________                                         RA = Rheumatoid Arthritis                                                     IDDM = Insulin dependent diabetes mellitus                               

If Felty's Disease patients or RA patients are further divided intosub-groups based upon their HLA DR and DW phenotypes, it is probablethat the elevation of δTCS1⁺ T cells will correlate even more stronglywith disease.

10.1.2. LONGITUDINAL STUDIES:

It is expected that the elevation of δTCS1⁺ T cells in RA or Felty'swill correlate with disease state (e.g., tender joints, vasculitis,etc.). In addition, it is well known that female RA patients undergoremission of symptoms shortly after becoming pregnant. Their symptomsreturn again after giving birth. Longitudinal studies are underway tostudy the levels of δTCS1⁺ T cells in RA and Felty's patients and inpregnant women. These numbers may then be correlated with the intensityof the disease during these stages. This data may be used to subdividepatients that would benefit from δTCS1 therapies designed to modulate(eliminate or block or stimulate or enhance) the δTCS1⁺ T cell subset.

10.1.3. SYNOVIAL FLUID AND SYNOVIUM TISSUE STUDY

Analysis of RA synovium-derived T cells upon expansion in IL2supplemented culture fluid and of synovial fluid T cells yieldedelevated levels of δTCS1⁺ cells in RA patients as was seen forperipheral blood (see Examples 8 & 9, supra). In many RA samples, pairedblood and synovium tissues from the same patients were studied (seeExample 9, supra). Furthermore, we further evaluated the δTCS1⁺ T cellsin RA synovium by using in situ immunohistochemistry (Example 8, supra).6 out of 11 RA patients studied showed significant infiltration ofδTCS1⁺ T cells in synovium. This indicated that the elevation of δTCS1was not due to a bias in cell growth during in vitro culture conditions.The detailed relationship between the δTCS1⁺ T cells in the blood andsynovium of the same RA patient is being investigated further.

10.1.4. ACTIVITY OF γδ⁺ T CELLS IN RHEUMATOID ARTHRITIS

The γδ⁺ cells from some RA synovial tissue derived T cell linespossessed cytotoxic activity against their autologous type Asynoviocytes, and other lines produced factors with suppressor activity.The antibody, δTCS1, was able, under varied conditions, to eitherenhance the cytotoxic activity, to mitogenically stimulate the cells, orto block the activity of the cells (Example 9, supra).

Taken together the data suggests that δTCS1⁺ T cells play a significantrole in the pathogenesis of rheumatoid arthritis patients and that itcan be used as a T cell receptor-specific therapeutic.

10.1.5. αβ T CELL ANALYSIS IN ARTHRITIS

Evidence has been generated that T cells infiltrating synovium of someRA patients primarily express Vβ3, Vβ9, and Vβ10 (Example 11, infra).These 3 Vβ's together account for 5% of total T cells in normalsubjects. This demonstrated that distinct subsets of T cells thatrepresent only a small fraction of the T cells in normal subjects may bepreferentially associated with some RA patients. Thus, it appears thatat least two distinct groups of RA patients can be determined based uponTCAR expression; the first group expresses Vβ3, Vβ9, or Vβ10 in thesynovium, while the second group expresses primarily Vδ1.

10.1.6. TOXICOLOGY OF δTCS1 MONOCLONAL ANTIBODY

The acute and chronic toxicity of δTCS1 antibody may be determined bystandard animal model procedures. However, data has been collected withother similar antibodies; e.g. OKT3. The documented information supportsthe general safety of mouse IgG_(2a) antibody administered to humansubjects in amounts up to several grams per person per day (ORTHOCLONEOKT3 (MUROMONAB-CD3) Product Insert, Ortho Pharmaceutical Corp.,Raritan, N.J.).

The question of possible cross reactivity of δTCS1 antibody with otherhuman tissues has been examined. It does not appear to react with any ofthe other blood cell types, such as neutrophils, monocytes and red bloodcells, nor with tissues in the gastrointestinal tracts. It does,however, weakly react with some Langerhans cells in the skin and someintracellular antigen in the glandular cells of endometrium.

Sensitization to a murine monoclonal antibody, such as OKT3, has beenobserved in most patients under treatment, but has not producedsignificant symptoms of hypersensitivity, anaphylaxis or serum sickness(ORHTOCLONE OKT3 (MUROMONAB-CD3) Product insert, Ortho PharmaceuticalCorp., Raritan, N.J.). Similar results may be expected for δTCS1.

Almost all of the patients treated with OKT3 developed an acute symptomcomplex with chills and fever after the first injection. This typicallycommenced 45-60 minutes after the antibody injection and lasted forseveral hours. This acute symptom was presumed to be due to aphysiological response to the rapid lysis of large numbers of T cellsduring the therapy (ORTHOCLONE (MUROMONAB-CD3 Product insert, supra).Since δTCS1 antibody will lyse a significantly smaller number of T cells(about 1%), the severity of the side effects associated with OXT3 shouldbe minimized.

10.1.7. PHARMACOKINETICS OF δTCS1 MONOCLONAL ANTIBODY

Data on the pharmacokinetics or pharmacology of δTCS1 antibody may bedetermined by standard animal model techniques. However, data on thisimportant information has been collected with other mouse monoclonalantibodies, such as OKT3, which may be applicable to δTCS1 antibody.

With a single bolus I.V. injection of 5 mg of OKT3 in a normal subject,the average half life of the antibody averages about 4 hours. Duringtreatment with 5 mg per day for 14 days, mean serum levels of the drugrose increased the first three days and then averaged 0.9 μg/ml on days3 to 14 (ORTHOCLONE (MUROMONAB-CD3) Product Insert, supra).

10.1.8. PHYSICAL BIOCHEMICAL PROPERTIES OF δTCS1 MONOCLONAL ANTIBODY

The δTCS1 monclonal antibody was generated by fusion of myeloma cellswith splenocytes derived from a BALB/c mouse immunized with a humanleukemic cell line, Molt 13. Detailed screening, cloning selection, andcharacterization procedures have been published (Wu, Y-J., et al., 1988,J. Immunol., 141, 1476-1479) and are incorporated by reference herein.

The antibody was initially produced as an IgG1 isotype. To improve theantibody dependent cytotoxicity (ADCC), which is the key in vivomechanism for cell elimination, an IgG_(2a) variant was selected asdescribed in Section 5.2, supra.

The parent IgG1 isotype and isotype switched IgG2a antibodies weretested by Ouchterloney diffusion, ELISA isotype test, and competitionassay. F(ab)₂ fragments were also generated and characterized bypolyacrylamide gel electrophoresis.

The physiochemical properties of the variant are identical to that ofthe IgGI parent in terms of mitogenic properties, cell reactivities andbinding. The IgG_(2a) δTCS1 antibody will be used as the therapeuticdrug for an elimination protocol.

10.2. CLINICAL PLAN FOR ELIMINATION PROTOCOL

Since one of the therapeutically important properties of δTCS1monoclonal antibody may be to target the specific δTCS1⁺ T cell subsetfor elimination, the following plan is given as a example of this typeof protocol.

10.2.1. INDICATION

The intended indication will be for use as adjunctive therapy fortreating RA patients who fail or are contraindicated for conventionalsecond line drugs.

10.2.2. PATIENT ADMISSION CRITERIA TO THE STUDY

RA patients who may be enrolled in the study include those who A)exhibit 5% or greater of δTCS1⁺ T cells in the periperal blood, and B)failed the conventional second line drugs. The screening will be basedon the immunofluorescense staining of potential patients' lymphocyteswith δTCS1 monoclonal antibody. It is estimated that at least 10 out of50 severe RA patients screened may be suitable for the study.

10.2.3. CLINICAL ENDPOINT

The initial clinical endpoint relates to a substantial reduction ofδTCS1⁺ T cells and possibly other T cell subsets in circulation within 2hours after the drug's administration. This will be carried out by theimmunofluorescence procedure described above.

The long-term clinical endpoint may be evaluated by clinicallymonitoring patients and lab indices approved by the American RheumatismAssociation (FIG. 17). The first signs of clinical improvements may comefrom the reduction of tender joint counts and duration of morningstiffness.

10.2.4. CALCULATION OF DRUG DOSE

A similar approach used for the calculation of OKT3 dose may beemployed. Assuming 1) there are 7 liters of blood in an adult, 2) thereare 1×10⁸ δTCS1⁺ T cells (10% of total T cells in RA) per liter of bloodin RA patients, 3) δTCS1⁺ T cells sequestered in lymphoid tissues equalsto that of blood, 4) it will require 15 μg of δTCS1⁺ monoclonal antibody(Ab) for 10⁸ T cells to induce effective cell elimination or block ofcell receptor function in vivo and 5) 5% of injected δTCS1⁺ antibody isbioavailable, then the calculated dose is 2.1 mg per dose (see below).##EQU2## 10.2.5. REGIMEN

The in vivo half life of mouse IgG_(2a) monoclonal antibody, known toone skilled in the art, averages around 4 hours. Therefore, a dailyinjection of about 2 mg per patient for 10 to 14 days is recommended inorder to achieve optimal efficiency.

10.3. SUMMARY

It is clear that lymphocytes are actively involved in the pathogenesisof Rheumatoid Arthritis (RA). Severe RA patients alone represent atleast a million people in the United States. Although immunosuppressiontherapies, such as total lymphoid irradiation, and thoracic ductdrainage, are efficacious for severe RA patients, but they are notsuitable for all patients. To date, no satisfactory immunotherapy isavailable for severe RA patients who have failed or contra-indicated forconventional second line drugs.

We have obtained evidence that about 1% of total T cells are involved insevere RA. An improved, novel, safer and efficacious immunotherapy ofselectively eliminating the same 1% of total T cells in the body, usingδTCS1 monoclonal antibody, has been developed. The best currentlyemployed experimental drug abolishes 60% of T cells, leading togeneralized immune suppression and raising serious issues relating topatient risk/benefit.

Studies of the infiltrated cells revealed that T cells represent themajor cell type at the site of tissue injury, that is, the synovium ofthe RA joints. These T cells have been the main target for otherimmunosuppressive drugs. Several independent studies indicate thatsynovial T cells are primarily CD4+ helper T cells. Our data suggeststhat these synovial T cells bear two distinct types of T cell antigenreceptors (TCR), namely, the αβ and the γδ TCR. Our evidence stronglysuggests that a subset of γδ cells, identifiable by the δTCS-1 antibody,is significantly elevated in the blood and/or synovium of about 20-30%of RA patients. These patients tend to develop more severe forms ofarthritis, i.e., Felty's Syndrome and RA with associated peripheralvasculitis. The lack of appropriate animal models, the historicalexcellent safety record of mouse monoclonal antibody products, and theseverity of RA disease leads us to believe that it is timely to proceedwith the drug development of the δTCS1 antibody as a useful therapeuticdrug for arthritis patients.

It has been noted that δTCS1⁺ T cells represent about 1% of total Tcells in healthy individuals. They are postulated to regulate thedevelopment of αβ T cells via the lymphokines they secrete and torepresent the motile population of γδ T cells in the body. The only FDAapproved mouse monoclonal antibody, therapeutic OKT3, eliminates 100% ofT cells, and the therapy requires supportive measures in a hospitalsetting for administration. However, with a 10-14 day course of dailyOKT3 injections, the therapeutic effects lasted for about one to twoyears in treated patients who suffered from transplanted kidneyrejection.

We propose to treat severe RA patients with δTCS1⁺ monoclonal antibodyby administering a daily I.V. dose of approximately 2 mg of the antibodyfor 10-14 days. The therapy is expected to be primarily maintained byoutpatient administration.

11. EXAMPLE: MONOCLONAL ANTIBODIES REACTIVE WITH THE VARIABLE REGIONS OFα,β HUMAN T CELL ANTIGEN RECEPTOR ARE USEFUL IN THE TREATMENT OFRHEUMATOID ARTHRITIS

The first step needed in the development of T cell receptor specifictherapeutics is to correlate specific T cell receptor gene usage withdisease. Once it is known which T cell receptors (TCRs) are primarilyinvolved in the disease, specific therapeutics can be produced.

A panel of TCR variable region genes were used to determine whichvariable regions correlate with rheumatoid arthritis. The data presentedinfra involves the analysis of rheumatoid arthritis patient samplesusing V.sub.α and V.sub.β TCR gene probes. Similar analysis could alsobe done using V.sub.γ and V.sub.δ genes as well.

11.1. MATERIALS AND METHODS

11.1.1. SAMPLES:

Paired synovial membrane derived T cell lines and peripheral blood Tcell lines were prepared from 12 patients with RA (see Example 9,infra). Peripheral blood lines were also obtained from 5 normalindividuals for controls using similar cell culture procedures.

11.1.2. T CELL RECEPTOR VARIABLE REGION GENE PROBES

There are 17 human V.sub.α and 18 human V.sub.β subfamilies that havebeen identified to date (Toyonaga, B. and Mak, T. W., 1987, Annual Rev.Immunol., 5, 585-620). These Vβ subfamilies are named Vβ1 to Vβ 20.Subfamilies designated Vβ13 and Vβ14 have been merged into otherfamilies based upon the degree of sequence homology of the members. Allexcept V.sub.α15 and V.sub.β16 have currently been tested. In addition,there are about 5-10 human V.sub.γ (Forster, A., et al., 1987, EMBO, 6,1945-1950) and 5-10 human V₆₇ (Takihara, Y., et al., 1989, J. Exp. Med.,169, 393) subfamilies that have been identified to date. As additional Vα, β, γ, and δ regions become available, they may similarly be tested.Once correlations between disease and specific TCR V subfamilies havebeen identified, the specific member of the subfamily responsible forthe correlation can also be identified (see infra).

11.1.3. RNA PREPARATIONS:

RNA was isolated by the guanidinium isothiocyanate cesium chlorideprocedure (Maniatis, T., et al., 1982, In "Molecular Cloning: ALaboratory Manual", Cold Spring Harbor Laboratories, N.Y.). Total RNAwas precipitated twice in 0.3 M sodium acetate and 2.5 volumes ofethanol. On average, 5 to 10 μg of total RNA was obtained from 10million cultured T cells.

11.1.4. T CELL ANTIGEN RECEPTOR USAGE ANALYSIS:

The usage of T cell antigen receptor α and β chains in the T cell lineswas determined using 3 major steps; i) cDNA synthesis; ii) polymerasechain reaction amplification; and iii) DNA slot blot analysis.

11.1.4.1. cDNA SYNTHESIS:

Five μg of total RNA from each sample was primed for cDNA synthesisusing the C.sub.α olignucleotide and a C.sub.β oligonucleotide. Toanalyze TCR γ,δ gene usage, C.sub.γ and C.sub.δ primers could be used inan analogous fashion. Both C.sub.α and C.sub.β primers were 18-merssynthesized by New England Biolabs, Beverly, Mass. using the publishedsequences of the α and β constant regions (Yanagi, Y., et al., 1984,Nature, 308, 145-149). The sequence of the C.sub.α primer(5'-TTAGAGTCTCTCAGCTGG-3') is located 31 nucleotides 3' from the NH₂terminus of the α chain constant region. The sequence for the C.sub.βprimer (5'-TTCTGATGGCTCAAACAC-3') is located 36 nucleotides 3' from theNH₂ terminus of the β chain constant region. The C.sub.β oligonucleotideprimed cDNA synthesis from both β chain constant regions (Yanagi, Y., etal., 1984, Nature, 308, 145-149; Jones, N., et al., 1985, Science, 227,311-314). The location of these primers was chosen such that thesynthesized cDNA would comprise the variable, diversity, and joiningregions of the T cell receptor mRNA and only a small portion of theconstant region.

First strand DNA synthesis was performed according to publishedprocedures (Okayama, H. and Berg, P., 1982, Mol. Cell. Biol., 2,161-170; Gubler, U. and Hoffman, B. J., 1983, Gene, 25, 263-269) exceptthat the reaction was terminated prior to synthesis of the secondstrand. The resulting templates were in the form of RNA:DNA hybrids.These duplexes were then used in an oligo-dG tailing reaction (Deng,G-R. and Wu, R., 1983, Meth, in Enzymol., 100, 96-117) whichpreferentially tails the 3' end of the cDNA strand over the RNA strand.

11.1.4.2. POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION:

The PCR reaction was performed in a thermocycler (Perkin-Elmer, Norwalk,Conn.) using recombinant Tag DNA polymerase (Cetus Corp., Emeryville,Calif.). Oligonucleotides, d(C)₁₀, and Cα and Cβ, were used as primersfor amplification. The PCR amplification procedure of Loh, E. Y., et al.(1989, Science, 243, 217-220) was used with the following modifications.PCR amplification was done for 30 cycles with each cycle comprisingincubations at 92° C. for 1 minute, 50° C. for 1.5 minutes and 72° C.for 2.5 minutes. The last extension reaction was for 10 minutes at 72°C. All samples were amplified a total of 3 times with isolation of theamplified DNA fragment of about 300-400 base pairs between each round.The final amplified DNA samples were then precipitated with spermine toremove free nucleotides, before labeling with ³² P radiolabelednucleotides. Labeling was done during 5 cycles of PCR amplificationusing all four ³² P labeled nucleotides at a ratio of 1:10non-radiolabeled nucleotides. The resulting ³² P labeled DNAs werepurified on elute-tip™ columns (Schleicher & Schuell, Keene, N.H.) toremove non-incorporated ³² P nucleotides.

11.1.4.3. DNA SLOT BLOT ANALYSIS:

DNA slot blots were prepared using a slot blot apparatus (Schleicher &Schuell, Keene, N.H.) and nylon membranes (Oncor, Gaithersburg, Md.)according to manufacturer's protocols. A panel of cDNA subclonescomprising the variable region of α and β chain TCR genes was spotted induplicate on each slot blot (3 μg per slot). After the blots had beenprepared containing the panel of TCR V region DNAs, individual blotswere then hybridized to the ³² P labeled T cell derived cDNA generatedin step #2. Individual patient samples were hybridized to duplicateblots. Hybridization condition and washes (Southern, E., 1979, J. Mol.Biol., 98, 503-517) were chosen to ensure no cross-hybridization betweenmembers of different subfamilies. The wash steps were performed at 42°C. in 0.2× SSC (30 mM sodium chloride, 3 mM sodium citrate, pH 7.4) with0.1% sodium dodecyl sulfate using 4 washes of 20 minutes each. Followingwashing, the blots were blotted dry, and then autoradiographed at -70°C. for 2-6 days using Eastman Kodak, X-Omat Xray film (Rochester, N.Y.).The developed autoradiographs were than scanned for intensity using avideo densitometer (Model 620, Biorad Corp., Richamond, Calif.).

11.2. RESULTS

Even in a normal disease free state, the expression of TCRs varies forthe different subfamilies. Some subfamilies, e.g. Vβ8, Vβ6 and Vα10, areexpressed quite frequently and the expression of others is fairly rare.For disease correlation, the increased levels of expression in diseaseare determined relative to these base levels.

Using the cDNA synthesis, PCR amplification and slot blot hybridizationprocedure detailed in the materials and methods, paired RA samplesincluding peripheral blood and synovial tissue derived T cell lines fromeach of 12 patients were analyzed relative to the expression in 5 normalperipheral blood controls. One basic assumption in this analysis is thatthe disease related T cells are most abundant at the site of thedisease, e.g. the synovial membrane of patients with rheumatoidarthritis.

An example of this analysis is shown in FIG. 18. The left panel of FIG.18, shows the autoradiograph obtained when the T cell line ST-2 obtainedfrom synovial tissue infiltrating lymphocytes was analyzed with thepanel of Vβ TCR genes. The right side of this figure shows thedensitometry trace. In this cell line, it is clear that several TCR Vβgenes (Vβ's 2, 4, 6, 7, 8, 11 and 18) are expressed with Vβ4 beingexpressed in highest amounts. To determine which of these correlate withdisease, this pattern of expression was compared to the pattern ofexpression observed in the peripheral blood derived T cell line (seeFIGS. 19 and 20).

FIG. 19 tabulates the results observed for Vβ gene expression in each ofthe paired synovium tissue and peripheral blood derived T cell linesfrom the 12 RA patients analyzed. The X axis represents the number ofpatient samples (12 total) where a Vβ was observed by the densitometryanalysis as illustrated in FIG. 18 for the ST-2 cell line. The Y axisrepresents each of the 16 Vβ gene probes tested. Peripheral blood datais represented by a crosshatched bar and synovial tissue data isrepresented by an open bar for each Vβ. From this figure, it can bedetermined that in the 12 RA patient samples analyzed, Vβ3, Vβ9, Vβ10and Vβ12 were expressed more often in the synovial tissue derived T celllines than in the peripheral blood derived T cell lines. For example,the ratio of presence in synovium to presence in the peripheral bloodsample was found to be 1.4 Vβ3. By this analysis, the most frequentlyexpressed Vβ genes in the synovium relative to the peripheral blood wereVβ3, Vβ9, Vβ10 and Vβ12.

When the same data was analyzed as shown in FIG. 20, the frequently usedgenes were Vβ1 (ratio=4.0), Vβ3 (ratio=infinity), Vβ6 (ratio=3.0), Vβ9(ratio=infinity), and Vβ10 (ration=infinity). For the analysis in FIG.20 only the dominant Vβ in each sample as determined by the desitometrytrace was used; the assumption being that although the T cell line maycontain varying subpopulations of T cells, the dominant subpopulationcould be the most relevant one. The frequencies of Vβ3, Vβ9, and Vβ10were high when the data from the 12 patients was analyzed either fortotal expression or dominant expression.

When the same samples were analyzed for total Vα gene expression (FIG.21), the results were less clear. The reason for this turned out to bethat 85% of the synovium or peripheral blood derived T cell linesanalyzed preferentially used Vα10 (FIG. 22). Although other Vα's werealso represented in the cell line populations. Vα10 was by far thedominant one with the densitometry peak height for Vα10 being 100 foldgreater than those of the other 15 Vα genes. This raises the possibilitythat Vα10 may represent a universal Vα that can pair well with most Vβchains. FIG. 21 shows that Vα12 (ratio=infinity) may be the next mostcommonly expressed Vα gene in synovium, but its level of expression islow when compared to the level of expression of Vα10.

11.3. SUMMARY

This analysis has shown that T cell populations at the site of disease,e.g. the joint synovial membrane, appear to predominantly expressspecific Vβ chains. One mechanism of autoimmunity may be thatdisease-related autoantigens are recognized by the body's own T cellsvia specific T cell antigen receptor α, β, γ and δ chains. After antigenrecognition, these T cells clonally expand to give rise to anoligoclonal population of disease-related T cells. Other mechanisms thatmay be involved include recruitment of specific cells to the diseasesite which would then represent an oligoclonal population of cells. Inthe total population of cells present at the disease site, theoligoclonal cells can be detected, as they will be using the TCRvariable regions that are most frequently expressed. To date, our studyhas shown that the most frequently expressed Vβ genes in the synovialmembrane of 12 RA patients were Vβ3, Vβ9, and Vβ10 and Vδ1 waspreferentially used in the γδ+ T cells present in synovial fluid. Torefine this correlation even more, patient HLA type, disease state andexpression of TCR genes for α, β, γ and δ chains and for TCRDiversity-Joining region expression may be determined. It is expectedthat as patients are subgrouped by HLA type, the disease correlationswill become even stronger.

11.4. DISCUSSION: TREATMENT OF RHEUMATOID ARTHRITIS PATIENTS WITH TCR α,β SPECIFIC REAGENTS

Once a disease correlation has been made between a disease state andspecific TCR gene expression, then the next step is to develop the TCRspecific therapeutics. One class of such therapeutics are anit-TCRantibodies.

For the analysis presented supra on the preferential use of 3 Vβ genesin rheumatoid arthritis patients, it is envisioned that a specifictherapeutic would involve a multiple antibody cocktail of anti-TCRantibodies specific for Vβ3, Vβ9 and Vβ10. This therapeutic would thustarget only the T cell subsets expressing these 3 Vβ TCRs and not effectother non-expressing T cells.

12. DEPOSIT OF HYBRIDOMAS

The following hybridoma cell lines, producing the indicated monoclonalantibody, have been deposited with the American Type Culture Collection,10801 University Blvd., Manassas, Va. 20110-2209, and have been assignedthe listed accession numbers:

    ______________________________________                                        Hybridoma  Monoclonal Antibody                                                                          Accession Number                                    ______________________________________                                        δTCAR-3                                                                            δTCS1 (δTCAR-3)                                                                  HB 9578                                               3A8 αF1 HB 9900                                                         3D6 αF2 HB 9901                                                         W112 6G-2 W112 HB 9927                                                        2D1 2D1 HB 9928                                                               5.A6.E9 Anti-TCRδ1 HB 9772                                              RS2A-2-H-7 δTCS1, isotype IgG2a HB 10110                              ______________________________________                                    

The present invention is not to be limited in scope by the cell linesdeposited since the deposited embodiments are intended as singleillustrations of one aspect of the invention and any cell lines whichare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A monoclonal antibody or derivative or fragmentthereof reactive with an epitope of the Vβ8.1 variable region of thebeta chain of a T cell antigen receptor.
 2. The monoclonal antibody, orderivative, or fragment of claim 1 wherein the antibody has theidentifying characteristics of monoclonal antibody 2D1, as produced bythe hybridoma deposited with the ATCC and assigned accession number HB9928.
 3. A hybridoma cell line which produces the monoclonal antibody ofclaim
 1. 4. A Fv, Fab, Fab', or F(ab')₂ fragment of the monoclonalantibody of claim
 1. 5. Monoclonal antibody 2D1, as produced by thehybridoma deposited with the ATCC and assigned accession number HB 9928.6. An antibody comprising the Fv, Fab, Fab', or F(ab')₂ fragment of themonoclonal antibody of claim
 5. 7. A hybridoma cell line, deposited withthe ATCC and assigned accession number HB 9928, which producesmonoclonal antibody 2D1.
 8. A method of treating a disease associatedwith abnormally high or low levels of a subset of T cells or with theinfiltration of a subset of T cells into a disease site wherein the Tcells of the subset express a V 8 variable region protein of a T cellantigen receptor, comprising administering in a suitable pharmaceuticalcarrier, to a patient an amount of a monoclonal antibody, or fragment orderivative thereof, effective to modulate a subset of T cells, whereinsaid monoclonal antibody recognizes an epitope of V 8.1 variable regionof a T cell antigen receptor.
 9. The method of claim 8, in which themonoclonal antibody or fragment or derivative thereof is 2D1, asproduced by the hybridoma deposited with the ATCC under accession numberHB 9928.